7 This resolution is limited by the shortest wavelength of light used to illuminate the specimen.

7 Light microscopes can magnify effectively to about 1,000 times the size of the actual specimen; at greater magnifications, the image becomes increasingly blurry.

7 Most of the improvements in light microscopy since the beginning of the 20th century have involved new methods for enhancing contrast, which clarifies the details that can be resolved (TABLE 7.1, p. 110).

7 In addition, scientists have developed methods for labeling particular cell components so that they stand out visually.

7 Fig 7-1. The size range of cells.

7 Most cells (yellow region of chart) are between 1 and 100 mm in diameter and are therefore visible only under a microscope.

7 Notice that the scale along the left side is logarithmic to accommodate the range of sizes shown.

7 Starting at the top of the scale with 10 m and going down, each reference measurement marks a tenfold decrease in diameter or length.

7 The fluorescing molecules may occur naturally in the specimen but more often are made by tagging the molecules of interest with fluorescent molecules.

7 Confocal. Uses lasers and special optics for "optical sectioning."

7 Only those regions within a narrow depth of focus are imaged.

7 Regions above and below the selected plane of view appear black rather than blurry.

7 This microscope is typically used with fluorescently stained specimens, as in the example here.

7 Although cells were discovered by Robert Hooke in 1665, the geography of the cell was largely uncharted until the 1950s.

7 Most subcellular structures, or organelles, are too small to be resolved by the light microscope.

7 Cell biology advanced rapidly in the 1950s with the introduction of the electron microscope.

7 Instead of using light, the electron microscope (EM) focuses a beam of electrons through the specimen or onto its surface.

7 Resolving power is inversely related to the wavelength of radiation a microscope uses, and electron beams have wavelengths much shorter than the wavelengths of visible light.

7 Modern electron microscopes can theoretically achieve a resolution of about 0.1 nanometer (nm), but the practical limit for biological structures is generally only about 2 nm--still a hundredfold improvement over the light microscope.

7 Biologists use the term cell ultrastructure to refer to a cell’s anatomy as revealed by an electron microscope.

7 There are two basic types of electron microscopes: the transmission electron microscope (TEM) and the scanning electron microscope (SEM).

7 Cell biologists use the TEM mainly to study the internal ultrastructure of cells.

7 The TEM aims an electron beam through a thin section of the specimen, similar to the way a light microscope transmits light through a slide.

7 However, instead of using glass lenses, the TEM uses electromagnets as lenses to focus and magnify the image by bending the paths of the electrons.

7 The image is ultimately focused onto a screen for viewing or onto photographic film.

7 To enhance contrast in the image, very thin sections of preserved cells are stained with atoms of heavy metals, which attach to certain cellular structures (FIGURE 7.2a).

7 Fig 7-2. Electron micrographs.

7 (a) This micrograph, taken with a transmission electron microscope (TEM), profiles a thin section of part of a cell from a rabbit trachea (windpipe), revealing its ultrastructure.

7 b) A scanning electron microscope (SEM) produced this three-dimensional image of the surface of the same type of cell.

7 Both micrographs show motile organelles called cilia.

7 Beating of the cilia that line the windpipe helps move inhaled debris upward toward the pharynx (throat).

7 The SEM is especially useful for detailed study of the surface of the specimen (FIGURE 7.2b).

7 The electron beam scans the surface of the sample, which is usually coated with a thin film of gold.

7 The beam excites electrons on the sample’s surface, and these secondary electrons are collected and focused onto a screen.

7 The result is an image of the topography of the specimen.

7 The SEM has great depth of field, which results in an image that appears three-dimensional.

7 Electron microscopes reveal many organelles that are impossible to resolve with the light microscope.

7 But the light microscope offers advantages, especially for the study of live cells.

7 A disadvantage of electron microscopy is that the methods used to prepare the specimen kill the cells.

7 Also, these methods may introduce artifacts, structural features seen in micrographs that do not exist in the living cell.

7 (Artifacts can occur in light microscopy, too.)

7 Microscopes are the most important tools of cytology, the study of cell structure.

7 But simply describing the diverse organelles within the cell reveals little about their function.

7 Modern cell biology developed from an integration of cytology with biochemistry, the study of the molecules and chemical processes of metabolism.

7 A biochemical approach called cell fractionation has been particularly important in cell biology.

7 Cell biologists can isolate organelles to study their functions

7 The goal of cell fractionation is to take cells apart, separating the major organelles so that their functions can be studied (FIGURE 7.3).

7 The instrument used to fractionate cells is the centrifuge, a merry-go-round for test tubes that can spin at various speeds.

7 The most powerful machines, called ultracentrifuges, can spin as fast as 130,000 revolutions per minute (rpm) and apply forces on particles of more than 1 million times the force of gravity (1,000,000 g).

7 Fig 7-3. Cell fractionation.

7 Disrupted cells are centrifuged at various speeds and durations to isolate (fractionate) components of different sizes.

7 By determining which cell fractions are associated with particular metabolic processes, researchers can tie those functions to certain organelles.

7 Fractionation begins with homogenization, the disruption of cells.

7 The objective is to break the cells apart without damaging their organelles.

7 Spinning the soupy homogenate in a centrifuge at low speed separates the parts of the cell into two fractions: the pellet, consisting of the larger, heavier structures that become packed at the bottom of the test tube; and the supernatant, consisting of the smaller, lighter parts of the cell suspended in the liquid above the pellet.

7 The supernatant is decanted into another tube and centrifuged again at a higher speed.

7 The process is repeated, increasing the speed with each step, collecting smaller and smaller components of the homogenized cells in successive pellets.

7 Cell fractionation enables the researcher to prepare specific components of cells in bulk quantity in order to study their composition and functions.

7 By following this approach, biologists have been able to assign various functions of the cell to the different organelles, a task that would be far more difficult with intact cells.

7 For example, one cellular fraction collected by centrifugation has enzymes that function in the metabolic process known as cellular respiration.

7 The electron microscope reveals this fraction to be very rich in the organelles called mitochondria.

7 This evidence helped cell biologists determine that mitochondria are the sites of cellular respiration.

7 Cytology and biochemistry complement each other in correlating cellular structure and function.

7 A PANORAMIC VIEW OF THE CELL

7 Prokaryotic and eukaryotic cells differ in size and complexity.

7 Internal membranes compartmentalize the functions of a eukaryotic cell.

7 Every organism is composed of one of two structurally different types of cells: prokaryotic cells or eukaryotic cells.

7 Only the bacteria and archaea have prokaryotic cells.

7 Protists, plants, fungi, and animals all have eukaryotic cells.

7 Prokaryotic and eukaryotic cells differ in size and complexity.

7 All cells have several basic features in common:

7 They are all bounded by a membrane, called a plasma membrane.

7 Within the membrane is a semifluid substance, cytosol, in which organelles are found.

7 All cells contain chromosomes, carrying genes in the form of DNA.

7 And all cells have ribosomes, tiny organelles that make proteins according to instructions from the genes.

7 A major difference between prokaryotic and eukaryotic cells, indicated by their names, is that the chromosomes of a eukaryotic cell are located in a membrane-enclosed organelle called the nucleus.

7 The word prokaryotic is from the Greek pro, before, and karyon, kernel, referring here to the nucleus.

7 In a prokaryotic cell (FIGURE 7.4), the DNA is concentrated in a region called the nucleoid, but no membrane separates this region from the rest of the cell.

7 Size is a general aspect of cell structure that relates to function.

7 The logistics of carrying out metabolism set limits on cell size.

7 At the lower limit, the smallest cells known are bacteria called mycoplasmas, which have diameters between 0.1 and 1.0 mm.

7 These are perhaps the smallest packages with enough DNA to program metabolism and enough enzymes and other cellular equipment to carry out the activities necessary for a cell to sustain itself and reproduce.

7 Most bacteria are 1-10 mm in diameter, a dimension about ten times bigger than that of mycoplasmas.

7 Using arbitrary units of length, we can calculate the cell’s surface area (in square units), volume (in cubic units), and surface-to-volume ratio.

7 A high surface-to-volume ratio facilitates the exchange of materials between a cell and its environment.

7 At the boundary of every cell, the plasma membrane functions as a selective barrier that allows sufficient passage of oxygen, nutrients, and wastes to service the entire volume of the cell (FIGURE 7.6).

7 For each square micrometer of membrane, only so much of a particular substance can cross per second.

7 Rates of chemical exchange with the extra cellular environment might be inadequate to maintain a cell with a very large cytoplasm.

7 The need for a surface sufficiently large to accommodate the volume helps explain the microscopic size of most cells.

7 Larger organisms do not generally have larger cells than smaller organisms--simply more cells.

7 Fig 7-6. The plasma membrane.

7 The plasma membrane and the membranes of organelles consist of a double layer (bilayer) of phospholipids with various proteins attached to or embedded in it.

7 The phospholipid tails in the interior of a membrane are hydrophobic; the phospholipid heads, the exterior proteins and parts of proteins, and any carbohydrate side chains are hydrophilic and in contact with the aqueous solution on either side of the membrane.

7 Carbohydrate side chains are found only on the outer surface of the plasma membrane.

7 The specific functions of a membrane depend on the kinds of phospholipids and proteins present.

7 Prokaryotic cells will be described in detail in Chapters 18 and 27, and the possible evolutionary relationships between prokaryotic and eukaryotic cells will be discussed in Chapter 28.

7 Most of the discussion of cell structure that follows in this chapter applies to eukaryotic cells.

7 Internal membranes compartmentalize the functions of a eukaryotic cell

7 In addition to the plasma membrane at its outer surface, a eukaryotic cell has extensive and elaborately arranged internal membranes, which partition the cell into compartments--the membranous organelles mentioned earlier.

7 These membranes also participate directly in the cell’s metabolism; many enzymes are built right into the membranes.

7 Because the cell’s compartments provide different local environments that facilitate specific metabolic functions, incompatible processes can go on simultaneously inside the same cell.

7 Membranes of various kinds are fundamental to the organization of the cell.

7 In general, biological membranes consist of a double layer of phospholipids and other lipids.

7 Embedded in this lipid bilayer or attached to its surfaces are diverse proteins (see FIGURE 7.6).

7 However, each type of membrane has a unique composition of lipids and proteins suited to that membrane’s specific functions.

7 For example, enzymes embedded in the membranes of the organelles called mitochondria function in cellular respiration.

7 Before continuing with this chapter, examine the overviews of eukaryotic cells in FIGURES 7.7 and 7.8 on these two pages.

7 These figures introduce the various organelles and provide a map of the cell for the detailed tour upon which we will now embark.

7 FIGURES 7.7 and 7.8 also contrast animal and plant cells.

7 As eukaryotic cells, they have much more in common than either has with any prokaryote.

7 As you will see, however, there are important differences between plant and animal cells.

7 THE NUCLEUS AND RIBOSOMES

7 The nucleus contains a eukaryotic cell’s genetic library

7 Ribosomes build a cell’s proteins

7 At the first stop of our detailed tour of the cell are two organelles involved in the genetic control of the cell: the nucleus, which houses most of the cell’s DNA, and the ribosomes, which use information from the DNA to make proteins.

7 The nucleus contains a eukaryotic cell’s genetic library

7 The nucleus contains most of the genes in the eukaryotic cell (some genes are located in mitochondria and chloroplasts).

7 It is generally the most conspicuous organelle in a eukaryotic cell, averaging about 5 mm in diameter.

7 The nuclear envelope encloses the nucleus (FIGURE 7.9), separating its contents from the cytoplasm.

7 Fig 7-9. The nucleus and its envelope.

7 Within the nucleus is chromatin, consisting of DNA and proteins.

7 When a cell prepares to divide, individual chromosomes become visible as the chromatin condenses.

7 The nucleolus functions in ribosome synthesis.

7 The nuclear envelope, which consists of two membranes separated by a narrow space, is perforated with pores and lined by a nuclear lamina.

7 The nuclear envelope is a double membrane.

7 The two membranes, each a lipid bilayer with associated proteins, are separated by a space of about 20-40 nm.

7 The envelope is perforated by pores that are about 100 nm in diameter.

7 At the lip of each pore, the inner and outer membranes of the nuclear envelope are fused.

7 An intricate protein structure called a pore complex lines each pore and regulates the entry and exit of certain large macromolecules and particles.

7 Except at the pores, the nuclear side of the envelope is lined by the nuclear lamina, a netlike array of protein filaments (intermediate filaments) that maintains the shape of the nucleus.

7 There is also much evidence for a nuclear matrix, a framework of fibers extending throughout the nuclear interior.

7 (We will examine possible functions of the nuclear lamina and matrix in Chapter 19.)

7 Within the nucleus, the DNA is organized along with proteins into a fibrous material called chromatin.

7 Stained chromatin usually appears through both light microscopes and electron microscopes as a diffuse mass.

7 As a cell prepares to divide (reproduce), however, the thin chromatin fibers coil up (condense), becoming thick enough to be discerned as separate structures called chromosomes.

7 Each eukaryotic species has a characteristic number of chromosomes.

7 A typical human cell, for example, has 46 chromosomes in its nucleus; the exceptions are the sex cells (eggs and sperm), which have only 23 chromosomes in humans.

7 A prominent structure within the nondividing nucleus is the nucleolus, which appears through the electron microscope as a mass of densely stained granules and fibers adjoining part of the chromatin.

7 Here a special type of RNA called ribosomal RNA is synthesized and assembled with proteins imported from the cytoplasm into the main components of ribosomes, called ribosomal subunits.

7 These subunits then pass through the nuclear pores to the cytoplasm, where they can combine to form ribosomes.

7 Sometimes there are two or more nucleoli; the number depends on the species and the stage in the cell’s reproductive cycle.

7 As we saw in FIGURE 5.28, the nucleus directs protein synthesis by synthesizing messenger RNA (mRNA) and sending it to the cytoplasm via the nuclear pores.

7 The mRNA is made according to instructions provided by the DNA (as is ribosomal RNA).

7 Once an mRNA molecule reaches the cytoplasm, ribosomes translate its genetic message into the primary structure of a specific polypeptide.

7 This process of translating genetic information is described in detail in Chapter 17.

7 Ribosomes build a cell’s proteins

7 Ribosomes, particles made of ribosomal RNA and protein, are the organelles that carry out protein synthesis; each is composed of two subunits (FIGURE 7.10).

7 Cells that have high rates of protein synthesis have a particularly large number of ribosomes.

7 For example, a human pancreas cell has a few million ribosomes.

7 Not surprisingly, cells active in protein synthesis also have prominent nucleoli.

7 (Keep in mind that both nucleoli and ribosomes, unlike most other organelles, are not enclosed in membrane.)

7 Fig 7-10. Ribosomes. (a) This electron micrograph of part of a pancreas cell shows many ribosomes, both free (in the cytosol) and bound (to the endoplasmic reticulum).

7 The bound ribosomes of pancreas cells make a number of secretory proteins, including the hormone insulin and digestive enzymes.

7 Bound ribosomes also make proteins destined for insertion into membranes or the interiors of other organelles.

7 Free ribosomes mainly make proteins that remain dissolved in the cytosol.

7 Bound and free ribosomes are identical and can alternate between these two roles.

7 Free ribosomes are suspended in the cytosol, while bound ribosomes are attached to the outside of the endoplasmic reticulum or nuclear envelope.

7 Most of the proteins made by free ribosomes will function within the cytosol; examples are enzymes that catalyze the first steps of sugar breakdown.

7 Bound ribosomes generally make proteins that are destined either for insertion into membranes, for packaging within certain organelles such as lysosomes, or for export from the cell (secretion).

7 Cells that specialize in protein secretion--for instance, the cells of the pancreas and other glands that secrete digestive enzymes--frequently have a high proportion of bound ribosomes.

7 Bound and free ribosomes are structurally identical and can alternate between the two roles; the cell adjusts the relative numbers of each as its metabolism changes.

7 You will learn more about ribosome structure and function in Chapter 17.

7 THE ENDOMEMBRANE SYSTEM

7 The endoplasmic reticulum manufactures membranes and performs many other biosynthetic functions

7 The Golgi apparatus finishes, sorts, and ships cell products

7 Lysosomes are digestive compartments\

7 Vacuoles have diverse functions in cell maintenance

7 Many of the different membranes of the eukaryotic cell are part of an endomembrane system.

7 These membranes are related either through direct physical continuity or by the transfer of membrane segments as tiny vesicles (sacs made of membrane).

7 Despite these relationships, the various membranes are not identical in structure and function.

7 Moreover, the thickness, molecular composition, and metabolic behavior of a membrane are not fixed, but may be modified several times during the membrane’s life.

7 The endomembrane system includes the nuclear envelope, endoplasmic reticulum, Golgi apparatus, lysosomes, various kinds of vacuoles, and the plasma membrane (not actually an endomembrane in physical location, but nevertheless related to the endoplasmic reticulum and other internal membranes).

7 We have already discussed the nuclear envelope and will now focus on the endoplasmic reticulum and the other endomembranes to which it gives rise.

7 The endoplasmic reticulum manufactures membranes and performs many other biosynthetic functions

7 The endoplasmic reticulum (ER) is a membranous labyrinth so extensive that it accounts for more than half the total membrane in many eukaryotic cells.

7 (The word endoplasmic means "within the cytoplasm," and reticulum is Latin for "little net.")

7 The ER consists of a network of membranous tubules and sacs called cisternae (Latin, cisterna, a reservoir for a liquid).

7 The ER membrane separates the internal compartment of the ER, called the cisternal space, from the cytosol.

7 And because the ER membrane is continuous with the nuclear envelope, the space between the two membranes of the envelope is continuous with the cisternal space of the ER (FIGURE 7.11).

7 Fig 7-11. Endoplasmic reticulum (ER).

7 A membranous system of interconnected tubules and flattened sacs called cisternae, the ER is also continuous with the nuclear envelope.

7 (The drawing is a cutaway view.)

7 The membrane of the ER encloses a compartment called the cisternal space.

7 Rough ER, which is studded on its outer surface with ribosomes, can be distinguished from smooth ER in the electron micrograph (TEM).

7 There are two distinct, though connected, regions of ER that differ in structure and function: smooth ER and rough ER.

7 Smooth ER is so named because its cytoplasmic surface lacks ribosomes.

7 Rough ER appears rough through the electron microscope because ribosomes stud the cytoplasmic surface of the membrane.

7 As already mentioned, ribosomes are also attached to the cytoplasmic side of the nuclear envelope’s outer membrane, which is confluent with rough ER.

7 Functions of Smooth ER

7 The smooth ER of various cell types functions in diverse metabolic processes, including synthesis of lipids, metabolism of carbohydrates, and detoxification of drugs and poisons.

7 Enzymes of the smooth ER are important to the synthesis of lipids, including oils, phospholipids, and steroids.

7 Among the steroids produced by the smooth ER in animal cells are the sex hormones of vertebrates and the various steroid hormones secreted by the adrenal glands.

7 The cells that actually synthesize and secrete these hormones--in the testes and ovaries, for example--are rich in smooth ER, a structural feature that fits the function of these cells.

7 Liver cells provide one example of the role of smooth ER in carbohydrate metabolism.

7 Liver cells store carbohydrate in the form of glycogen, a polysaccharide.

7 The hydrolysis of glycogen leads to the release of glucose from the liver cells, which is important in the regulation of sugar concentration in the blood.

7 However, the first product of glycogen hydrolysis is glucose phosphate, an ionic form of the sugar that cannot exit the cell and enter the blood.

7 An enzyme embedded in the membrane of the liver cell’s smooth ER removes the phosphate from the glucose, which can then leave the cell.

7 Enzymes of the smooth ER help detoxify drugs and poisons, especially in liver cells.

7 Detoxification usually involves adding hydroxyl groups to drugs, making them more soluble and easier to flush from the body.

7 The sedative phenobarbital and other barbiturates are examples of drugs metabolized in this manner by smooth ER in liver cells.

7 In fact, barbiturates, alcohol, and many other drugs induce the proliferation of smooth ER and its associated detoxification enzymes.

7 This, in turn, increases tolerance to the drugs, meaning that higher doses are required to achieve a particular effect, such as sedation.

7 Also, because some of the detoxification enzymes have relatively broad action, the proliferation of smooth ER in response to one drug can increase tolerance to other drugs as well.

7 Barbiturate abuse, for example, may decrease the effectiveness of certain antibiotics and other useful drugs

7 The membrane of each cisterna in a stack separates its internal space from the cytosol.

7 Vesicles concentrated in the vicinity of the Golgi apparatus are engaged in the transfer of material between the Golgi and other structures.

7 Fig 7-12. The Golgi apparatus.

7 The Golgi apparatus consists of stacks of flattened sacs, or cisternae, which, unlike ER cisternae, are not physically connected.

7 (The drawing is a cutaway view.)

7 A Golgi stack receives and dispatches transport vesicles and the products they contain.

7 Materials received from the ER are modified and stored in the Golgi and eventually shipped to the cell surface or other destinations.

7 Note the vesicles joining and leaving the cisternae.

7 A Golgi stack has a structural and functional polarity, with a cis face that receives vesicles containing ER products and a trans face that dispatches vesicles (at right, TEM).

7 A Golgi stack has a distinct polarity, with the membranes of cisternae at opposite ends of the stack differing in thickness and molecular composition.

7 The two poles of a Golgi stack are referred to as the cis face and the trans face; these act, respectively, as the receiving and shipping departments of the Golgi apparatus.

7 The cis face is usually located near the ER.

7 Transport vesicles move material from the ER to the Golgi.

7 A vesicle that buds from the ER will add its membrane and the contents of its lumen (cavity) to the cis face by fusing with a Golgi membrane.

7 The trans face gives rise to vesicles, which pinch off and travel to other sites.

7 Products of the ER are usually modified during their transit from the cis pole to the trans pole of the Golgi.

7 Proteins and phospholipids of membranes may be altered.

7 For example, various Golgi enzymes modify the oligosaccharide portions of glycoproteins.

7 Oligosaccharides are first added to proteins in the rough ER, often during the process of polypeptide synthesis.

7 The resulting glycoprotein is then modified as it passes through the rest of the ER and the Golgi.

7 The Golgi removes some sugar monomers and substitutes others, producing a large variety of oligosaccharides.

7 In addition to its finishing work, the Golgi apparatus manufactures certain macromolecules by itself.

7 Many polysaccharides secreted by cells are Golgi products, including pectins and certain other noncellulose polysaccharides made by plant cells and incorporated along with cellulose into their cell walls.

7 Cellulose is made by enzymes located within the plasma membrane, which directly deposit this polysaccharide on the outside surface.

7 Golgi products that will be secreted depart from the trans face of the Golgi inside transport vesicles that eventually fuse with the plasma membrane.

7 The Golgi manufactures and refines its products in stages, with different cisternae between the cis and trans ends containing unique teams of enzymes.

7 Products in various stages of processing seem to be transferred from one cisterna to the next by vesicles.

7 Before a Golgi stack dispatches its products by budding vesicles from the trans face, it sorts these products and targets them for various parts of the cell.

7 Molecular identification tags, such as phosphate groups that have been added to the Golgi products, aid in sorting.

7 Finally, transport vesicles budded from the Golgi may have external molecules on their membranes that recognize "docking sites" on the surface of specific organelles or on the plasma membrane.

7 Lysosomes are digestive compartments

7 A lysosome is a membrane-bounded sac of hydrolytic enzymes that the cell uses to digest macromolecules (FIGURE 7.13).

7 There are lysosomal enzymes that can hydrolyze proteins, polysaccharides, fats, and nucleic acids--all the major classes of macromolecules.

7 These enzymes work best in an acidic environment, at about pH 5.

7 The lysosomal membrane maintains this low internal pH by pumping hydrogen ions from the cytosol into the lumen of the lysosome.

7 If a lysosome breaks open or leaks its contents, the released enzymes are not very active, because the cytosol has a neutral pH.

7 However, excessive leakage from a large number of lysosomes can destroy a cell by autodigestion.

7 From this example we can see once again how important compartmental organization is to the functions of the cell:

7 The lysosome provides a space where the cell can digest macromolecules safely, without the general destruction that would occur if hydrolytic enzymes roamed at large.

7 Fig 7-13. Lysosomes.

7 a) In this white blood cell from a rat, the lysosomes are very dark because of a specific stain that reacts with one of the products of digestion within the lysosome.

7 This type of white blood cell ingests bacteria and viruses and destroys them in the lysosomes (TEM).

7 (b) In the cytoplasm of this rat liver cell, an autophagic lysosome has engulfed two disabled organelles, a mitochondrion and a peroxisome (TEM).

7 Hydrolytic enzymes and lysosomal membrane are made by rough ER and then transferred to the Golgi apparatus for further processing.

7 At least some lysosomes probably arise by budding from the trans face of the Golgi apparatus (FIGURE 7.14, p. 122).

7 Proteins of the inner surface of the lysosomal membrane and the digestive enzymes themselves are probably spared from destruction by having three-dimensional conformations that protect vulnerable bonds from enzymatic attack.

7 Fig 7-14. The formation and functions of lysosomes.

7 The ER and Golgi apparatus generally cooperate in the production of lysosomes containing active hydrolytic enzymes.

7 Lysosomes digest (hydrolyze) materials taken into the cell and recycle materials from intracellular refuse.

7 This figure shows one lysosome fusing with a food vacuole and another engulfing a damaged mitochondrion.

7 Lysosomes carry out intracellular digestion in a variety of circumstances.

7 Amoebas and many other protists eat by engulfing smaller organisms or other food particles, a process called phagocytosis (from the Greek phagein, to eat, and kytos, vessel, referring here to the cell).

7 The food vacuole formed in this way then fuses with a lysosome, whose enzymes digest the food (see FIGURE 7.14).

7 Digestion products, including simple sugars, amino acids, and other monomers, pass into the cytosol and become nutrients for the cell.

7 Some human cells also carry out phagocytosis.

7 Among them are macrophages, cells that help defend the body by destroying bacteria and other invaders (see FIGURE 7.13a).

7 Lysosomes also use their hydrolytic enzymes to recycle the cell’s own organic material, a process called autophagy.

7 This occurs when a lysosome engulfs another organelle or a small amount of cytosol (see FIGURE 7.13b).

7 The lysosomal enzymes dismantle the ingested material, and the organic monomers are returned to the cytosol for reuse.

7 With the help of lysosomes, the cell continually renews itself.

7 A human liver cell, for example, recycles half of its macromolecules each week.

7 Programmed destruction of cells by their own lysosomal enzymes is important in the development of many multicellular organisms.

7 During the transforming of a tadpole into a frog, for instance, lysosomes in the cells of the tail destroy these cells.

7 And the hands of human embryos are webbed until lysosomes digest the tissue between the fingers.

7 Mitochondria and chloroplasts are the main energy transformers of cells\

7 Organisms are open systems that transform the energy they acquire from their surroundings

7 In eukaryotic cells, mitochondria and chloroplasts are the organelles that convert energy to forms that cells can use for work.

7 Mitochondria (singular, mitochondrion) are the sites of cellular respiration, the catabolic process that generates ATP by extracting energy from sugars, fats, and other fuels with the help of oxygen.

7 Chloroplasts, found only in plants and algae, are the sites of photosynthesis.

7 They convert solar energy to chemical energy by absorbing sunlight and using it to drive the synthesis of organic compounds from carbon dioxide and water.

7 Although mitochondria and chloroplasts are enclosed by membranes, they are not part of the endomembrane system.

7 Their membrane proteins are made not by the ER, but by free ribosomes in the cytosol and by ribosomes contained within the mitochondria and chloroplasts themselves.

7 Not only do these organelles have ribosomes, but they also contain a small amount of DNA.

7 It is this DNA that programs the synthesis of the proteins made on the organelle’s own ribosomes. (Proteins imported from the cytosol--constituting most of the organelle’s proteins--are programmed by nuclear DNA.)

7 Mitochondria and chloroplasts are semiautonomous organelles that grow and reproduce within the cell.

7 In Chapters 9 and 10, we will focus on how mitochondria and chloroplasts function.

7 We will consider the evolution of these organelles in Chapter 28.

7 Here we are concerned mainly with the structure of these energy transformers.

7 Mitochondria are found in nearly all eukaryotic cells, including those of plants, animals, fungi, and protists.

7 Some cells have a single large mitochondrion, but more often a cell has hundreds or even thousands of mitochondria; the number is correlated with the cell’s level of metabolic activity.

7 Mitochondria are about 1-10 mm long.

7 Time-lapse films of living cells reveal mitochondria moving around, changing their shapes, and dividing in two, unlike the static cylinders seen in electron micrographs of dead cells.

7 The mitochondrion is enclosed by two membranes, each a phospholipid bilayer with a unique collection of embedded proteins (FIGURE 7.17).

7 The outer membrane is smooth, but the inner membrane is convoluted, with infoldings called cristae.

7 The inner membrane divides the mitochondrion into two internal compartments.

7 The first is the intermembrane space, the narrow region between the inner and outer membranes.

7 The second compartment, the mitochondrial matrix, is enclosed by the inner membrane.

7 The matrix contains many different enzymes as well as the mitochondrial DNA and ribosomes.

7 Some of the metabolic steps of cellular respiration are catalyzed by enzymes in the matrix.

7 Other proteins that function in respiration, including the enzyme that makes ATP, are built into the inner membrane.

7 The cristae give the inner mitochondrial membrane a large surface area that enhances the productivity of cellular respiration, another example of structure fitting function.

7 Fig 7-17. The mitochondrion, site of cellular respiration.

7 The two membranes of the mitochondrion are evident in the drawing and micrograph (TEM).

7 The cristae are infoldings of the inner membrane.

7 The cutaway drawing shows the two compartments bounded by the membranes: the intermembrane space and the mitochondrial matrix.

7 The chloroplast is a specialized member of a family of closely related plant organelles called plastids.

7 The peroxisome is a specialized metabolic compartment bounded by a single membrane (FIGURE 7.19).

7 Peroxisomes contain enzymes that transfer hydrogen from various substrates to oxygen, producing hydrogen peroxide (H2O2) as a by-product, from which the organelle derives its name.

7 These reactions may have many different functions.

7 Some peroxisomes use oxygen to break fatty acids down into smaller molecules that can then be transported to mitochondria as fuel for cellular respiration.

7 Peroxisomes in the liver detoxify alcohol and other harmful compounds by transferring hydrogen from the poisons to oxygen.

7 The H2O2 formed by peroxisome metabolism is itself toxic, but the organelle contains an enzyme that converts the H2O2 to water.

7 Enclosing in the same space both the enzymes that produce hydrogen peroxide and those that dispose of this toxic compound is another example of how the cell’s compartmental structure is crucial to its functions.

7 Fig 7-19. Peroxisomes.

7 Peroxisomes are roughly spherical and often have a granular or crystalline core that is probably a dense collection of enzyme molecules.

7 Specialized peroxisomes called glyoxysomes are found in the fat-storing tissues of plant seeds.

7 These organelles contain enzymes that initiate the conversion of fatty acids to sugar, which the emerging seedling can use as an energy and carbon source until it is able to produce its own sugar by photosynthesis.

7 Unlike lysosomes, peroxisomes do not bud from the endomembrane system.

7 They grow by incorporating proteins and lipids made in the cytosol, and they increase in number by splitting in two when they reach a certain size.

12 In fact, the mitotic (M) phase, which includes both mitosis and cytokinesis, is usually the shortest part of the cell cycle.

12 Mitotic cell division alternates with a much longer interphase, which often accounts for about 90% of the cycle.

12 It is during interphase that the cell grows and copies its chromosomes in preparation for cell division.

12 Interphase can be divided into sub phases: the G1phase ("first gap"), the S phase, and the G2phase ("second gap").

12 During all three sub phases, the cell grows by producing proteins and cytoplasmic organelles.

12 However, chromosomes are duplicated only during the S phase (S stands for synthesis of DNA).

12 Thus, a cell grows (G1), continues to grow as it copies its chromosomes (S), grows more as it completes preparations for cell division (G2), and divides (M).

12 The first part of interphase, called G1, is followed by the S phase, when the chromosomes replicate; the last part of interphase is called G2.

12 In the M phase, mitosis divides the nucleus and distributes its chromosomes to the daughter nuclei, and cytokinesis divides the cytoplasm, producing two daughter cells.

12 Time-lapse films of living, dividing cells reveal the dynamics of mitosis as a continuum of changes.

12 For purposes of description, however, mitosis is conventionally broken down into five sub phases: prophase, prometaphase, metaphase, anaphase, and telophase.

12 FIGURE 12.5, on pages 218-219, describes these stages in an animal cell.

12 Be sure to study this figure thoroughly before progressing to the next section, which examines mitosis more closely.

12 Fig 12-5. The stages of mitotic cell division in an animal cell.

12 The light micrographs show dividing lung cells from a newt, which has 22 chromosomes in its somatic cells.

12 The chromosomes appear blue and the microtubules green. (The red fibers are intermediate filaments.)

12 The schematic drawings show details not visible in the micrographs.

12 For the sake of simplicity, only four chromosomes are drawn. (In plant cells, centrioles are lacking and cytokinesis occurs differently.

12 Many of the events of mitosis depend on the mitotic spindle, which begins to form in the cytoplasm during prophase.

12 This structure consists of fibers made of microtubules and associated proteins.

12 While the mitotic spindle assembles, the microtubules of the cytoskeleton partially disassemble, probably providing the material used to construct the spindle.

12 The spindle microtubules elongate by incorporating more subunits of the protein tubulin (see TABLE 7.2).

12 The assembly of spindle microtubules starts in the centrosome, a non-membranous organelle that functions throughout the cell cycle to organize the cell’s microtubules (it is also called the microtubule-organizing center).

12 In animal cells, a pair of centrioles is located at the center of the centrosome, but the centrioles are not essential for cell division.

12 In fact, the centrosomes of most plants lack centrioles, and if a researcher destroys the centrioles of an animal cell with a laser micro beam, a spindle nevertheless forms during mitosis.

12 During interphase, the single centrosome replicates to form two centrosomes (see FIGURE 12.5)

12 As mitosis starts, the two centrosomes are located near the nucleus; they then move apart from each other during prophase and prometaphase, as spindle microtubules grow out from them.

12 By the end of prometaphase, the two centrosomes, referred to as spindle poles in this context, are at opposite poles of the cell.

12 Each of the two joined chromatids of a chromosome has a kinetochore, a structure of proteins and specific sections of chromosomal DNA at the centromere.

12 The chromosome’s two kinetochores face in opposite directions.

12 During prometaphase, some of the spindle microtubules attach to the kinetochores.

12 When one of a chromosome’s kinetochores is "captured" by microtubules, the chromosome begins to move toward the pole from which those microtubules come.

12 However, this movement is checked as soon as microtubules from the opposite pole attach to the other kinetochore.

12 What happens next is like a tug-of-war that ends in a draw.

12 The chromosome moves first in one direction, then the other, back and forth, finally settling midway between the two poles of the cell (FIGURE 12.6).

12 Meanwhile, microtubules that do not attach to kinetochores interact with nonkinetochore microtubules from the opposite pole of the cell.

12 At metaphase, these microtubules overlap, and the centromeres of all the duplicated chromosomes are on a plane midway between the two poles.

12 The spindle is now complete.

12 Let’s now see how the structure of the completed spindle correlates with its function during anaphase.

12 Anaphase commences suddenly when proteins holding together the sister chromatids of each chromosome are inactivated.

12 Now that the chromatids are separate, full-fledged chromosomes, they move toward opposite poles of the cell.

12 How do the kinetochore microtubules function in this poleward movement of chromosomes?

12 Experimental evidence supports the hypothesis that kinetochores are equipped with motor proteins that "walk" a chromosome along the attached microtubules toward the nearest pole.

12 Meanwhile, the microtubules shorten by depolymerizing at their kinetochore ends (FIGURE 12.7). (To review how motor proteins move an object along a microtubule, see FIGURE 7.21b.)

12 What is the function of the nonkinetochore microtubules?

12 In a dividing animal cell, these microtubules are responsible for elongating the whole cell during anaphase.

12 Nonkinetochore microtubules interdigitate across the metaphase plate, and during anaphase ones originating from opposite spindle poles move past each other toward their poles.

12 The mechanism seems to be similar to the one that slides neighboring microtubules in a flagellum

12 Motor proteins attached to the nonkinetochore microtubules drive them past one another, using energy from ATP

12 At the same time, the nonkinetochore microtubules lengthen by the addition of tubulin subunits to their ends.

12 At the end of anaphase, duplicate sets of chromosomes have arrived at opposite poles of the elongated parent cell.

12 Nuclei re-form during telophase.

12 Cytokinesis generally begins during this last stage of mitosis.

12 In animal cells, cytokinesis occurs by a process known as cleavage.

12 The first sign of cleavage is the appearance of a cleavage furrow, which begins as a shallow groove in the cell surface near the old metaphase plate.

12 On the cytoplasmic side of the furrow is a contractile ring of actin microfilaments associated with molecules of the protein myosin.

12 Actin and myosin are the same proteins responsible for muscle contraction, as well as many other kinds of cell movement.

12 The contraction of the dividing cell’s ring of microfilaments is like the pulling of drawstrings.

12 The cleavage furrow deepens until the parent cell is pinched in two, producing two completely separated cells.

12 Cytokinesis in plant cells, which have walls, is markedly different.

12 There is no cleavage furrow.

12 Instead, during telophase, vesicles derived from the Golgi apparatus move along microtubules to the middle of the cell, where they coalesce, producing a cell plate.

12 Cell wall materials carried in the vesicles collect in the cell plate as it grows.

12 The cell plate enlarges until its surrounding membrane fuses with the plasma membrane along the perimeter of the cell.

12 Two daughter cells result, each with its own plasma membrane.

12 Meanwhile, a new cell wall arising from the contents of the cell plate has formed between the daughter cells.

12 Most bacterial genes are carried on a single bacterial chromosome that consists of a circular DNA molecule and associated proteins.

12 Although bacteria are smaller and simpler than eukaryotic cells, the problem of replicating their genomes in an orderly fashion and distributing the copies equally to two daughter cells is still formidable.

12 The chromosome of the bacterium Escherichia coli, for example, when it is fully stretched out, is about 500 times longer than the length of the cell.

12 Clearly, such a long chromosome must be highly coiled and folded within the cell--and it is.

12 Prokaryotes do not have mitotic spindles, so what brings about the separation of the two daughter chromosomes in a dividing bacterial cell?

12 A hypothesis proposed in the 1960s suggested that separation of bacterial chromosomes results simply from the growth of new plasma membrane between two sites on the membrane where the chromosome copies are attached.

12 Rather than being a passive process, separation of daughter bacterial chromosomes involves active chromosomal movement.

12 Once the DNA of the chromosome begins to replicate, the copies of the first replicated region--called the origin of replication--move apart rapidly.

12 Using the techniques of modern DNA technology to tag the origins of replication with molecules that glow green in fluorescence microscopy, Researchers have directly observed the movement of bacterial chromosomes.

12 This movement is reminiscent of the poleward movements of the centromere regions of eukaryotic chromosomes during anaphase of mitosis, even though bacteria don’t have mitotic spindles or even microtubules.

12 How bacterial chromosomes move is still a mystery.

12 The idea that prokaryotes might have molecules in any way like the microtubules and motor proteins used in eukaryotic mitosis is surprising and intriguing.

12 When chromosomal replication is complete, the plasma membrane grows inward to divide the cell in two as a new cell wall is deposited between the daughter cells.

12 The example shown here is the bacterium Escherichia coli

12 While the bacterial chromosome is replicating, the cell is growing.

12 When replication is complete and the bacterium has reached about twice its initial size, its plasma membrane grows inward, dividing the parent cell into two daughter cells.

12 Each cell inherits a complete genome.

12 As eukaryotes evolved, along with their larger genomes and nuclear envelopes, the ancestral process of binary fission somehow gave rise to mitosis.

12 FIGURE 12.11 traces a hypothesis for the stepwise evolution of mitosis.

12 Possible intermediate stages are represented by two unusual types of nuclear division found in certain modern unicellular algae.

12 In both types, the nuclear envelope remains intact.

12 In dinoflagellates, replicated chromosomes are attached to the nuclear envelope and separate as it elongates prior to cell division.

12 In diatoms, a spindle within the nucleus separates the chromosomes.

12 Fig 12-11. A hypothesis for the evolution of mitosis.

12 Researchers interested in the evolution of eukaryotic cell division have observed in modern organisms what they believe are mechanisms of division intermediate between the binary fission of bacteria and mitosis as it occurs in most eukaryotes.

12 These schematic diagrams of a proposed evolutionary sequence do not show cell walls.

13 In humans, each somatic cell--any cell other than a sperm or ovum--has 46 chromosomes.

13 With a light microscope, condensed (mitotic) chromosomes can be distinguished from one another by their appearance.

13 The sizes of chromosomes and the positions of their centromeres differ.

13 When chromosomes are stained with certain dyes, each chromosome also has a distinctive pattern of colored bands.

13 Careful examination of a micrograph of the 46 human chromosomes reveals that there are two of each type.

13 This becomes clear when images of the chromosomes are arranged in pairs, starting with the longest chromosomes.

13 The resulting display is called a karyotype (FIGURE 13.3).

13 The chromosomes that make up a pair--that have the same length, centromere position, and staining pattern--are called homologous chromosomes, or homologues.

13 The two chromosomes of each pair carry genes controlling the same inherited characters.

13 For example, if a gene for eye color is situated at a particular locus on a certain chromosome, then the homologue of that chromosome will also have a gene specifying eye color at the equivalent locus.

13 Fig 13-3. Preparation of a human karyotype.

13 Karyotypes, ordered displays of an individual’s chromosomes, are often prepared using lymphocytes, a type of white blood cell.

13 The cells are treated with a drug to stimulate mitosis and are then grown in culture for several days.

13 Another drug is then added to arrest mitosis at metaphase, when the chromosomes, each consisting of two joined sister chromatids, are highly condensed and easy to identify in the microscope.

13 The figure above outlines the further steps in the preparation of the karyotype.

13 Karyotyping can be used to screen for abnormal numbers of chromosomes or defective chromosomes associated with certain congenital disorders, such as Down syndrome.

13 The causes and effects of chromosomal disorders are discussed in Chapter 15

13 There is an important exception to the rule of homologous chromosomes for human somatic cells: the two distinct chromosomes referred to as X and Y.

13 Human females have a homologous pair of X chromosomes (XX), but males have one X and one Y chromosome (XY).

13 Only small parts of the X and Y are homologous; most of the genes carried on the X chromosome do not have counterparts on the tiny Y, and the Y has genes lacking on the X.

13 Because they determine an individual’s sex, the X and Y chromosomes are called sex chromosomes.

13 The occurrence of homologous pairs of chromosomes in our karyotype is a consequence of our sexual origins.

13 We inherit one chromosome of each pair from each parent.

13 So the 46 chromosomes in our somatic cells are actually two sets of 23 chromosomes--a maternal set (from our mother) and a paternal set (from our father).

13 Sperm cells and ova are different from somatic cells in chromosome count.

13 Each of these reproductive cells, or gametes, has a single set of the 22 autosomes plus a single sex chromosome, either X or Y.

13 For humans, the haploid number, abbreviated n, is 23 (n = 23).

13 By means of sexual intercourse, a haploid sperm cell from the father reaches and fuses with a haploid ovum of the mother.

13 This union of gametes is called fertilization, or syngamy.

13 The resulting fertilized egg, or zygote, contains the two haploid sets of chromosomes bearing genes representing the maternal and paternal family lines.

13 The zygote and all other cells having two sets of chromosomes are called diploid cells.

13 For humans, the diploid number of chromosomes, abbreviated 2n, is 46 (2n = 46).

13 As a human develops from a zygote to a sexually mature adult, the zygote’s genes are passed on with precision to all somatic cells of the body by the process of mitosis.

13 Thus, somatic cells, like the zygote from which they are derived, are diploid.

13 The only cells of the human body not produced by mitosis are the gametes, which develop in the gonads (ovaries in females and testes in males).

13 Imagine what would happen if human gametes were made by mitosis: They would be diploid like the somatic cells.

13 At the next round of fertilization, when two gametes fused, the normal chromosome number of 46 would double to 92, and each subsequent generation would double the number of chromosomes yet again.

13 But sexually reproducing organisms carry out a process that halves the chromosome number in the gametes, compensating for the doubling that occurs at fertilization.

13 This process is a form of cell division called meiosis, and in animals it occurs only in the ovaries or testes.

13 While mitosis conserves chromosome number, meiosis reduces the chromosome number by half.

13 As a result, human sperm and ova each have a haploid set of 23 different chromosomes, one from each homologous pair.

13 Fertilization restores the diploid condition by combining two haploid sets of chromosomes, and the human life cycle is repeated, generation after generation (FIGURE 13.4).

13 Fig 13-4. The human life cycle.

13 In each generation, the doubling of chromosome number that results from fertilization is offset by the halving of chromosome number that results from meiosis.

13 For humans, the number of chromosomes in a haploid cell is 23 (n = 23); the number of chromosomes in the diploid zygote and all somatic cells arising from it is 46 (2n = 46).

13 In general outline, the human life cycle is typical of many animals.

13 Indeed, the processes of fertilization and meiosis are the unique trademarks of sexual reproduction.

13 Fertilization and meiosis alternate in sexual life cycles, offsetting each other’s effects on the chromosome number and thus perpetuating a species’ chromosome count.

13 Although the alternation of meiosis and fertilization is common to all organisms that reproduce sexually, the timing of these two events in the life cycle varies, depending on the species.

13 These variations can be grouped into three main types of life cycles (FIGURE 13.5).

13 The human life cycle is an example of one type, characteristic of most animals.

13 Gametes are the only haploid cells.

13 Meiosis occurs during the production of gametes, which undergo no further cell division prior to fertilization.

13 The diploid zygote divides by mitosis, producing a multicellular organism that is diploid (FIGURE 13.5a).

13 Fig 13-5. Three sexual life cycles differing in the timing of meiosis and fertilization (syngamy).

13 The common feature of all three cycles is the alternation of these two key events, which contribute to genetic variation among offspring.

13 A second type of life cycle occurs in most fungi and some protists, including some algae.

13 After gametes fuse to form a diploid zygote, meiosis occurs before offspring develop.

13 This meiosis produces not gametes but haploid cells that then divide by mitosis to give rise to a haploid multicellular adult organism.

13 Subsequently, the haploid organism produces gametes by mitosis, rather than by meiosis.

13 The only diploid stage is the zygote (FIGURE 13.5b).

13 Note that either haploid or diploid cells can divide by mitosis, depending on the type of life cycle.

13 Only diploid cells, however, can undergo meiosis.

13 Plants and some species of algae exhibit a third type of life cycle called alternation of generations.

13 This type of life cycle includes both diploid and haploid multicellular stages.

13 The multicellular diploid stage is called the sporophyte.

13 The multicellular diploid stage is called the sporophyte.

13 Meiosis in the sporophyte produces haploid cells called spores.

13 Unlike a gamete, a spore gives rise to a multicellular individual without fusing with another cell.

13 A spore divides mitotically to generate a multicellular haploid stage called the gametophyte.

13 The haploid gametophyte makes gametes by mitosis.

13 Fertilization results in a diploid zygote, which develops into the next sporophyte generation.

13 In this type of life cycle, therefore, the sporophyte and gametophyte generations take turns reproducing each other (FIGURE 13.5c).

13 Though the three types of sexual life cycles differ in the timing of meiosis and fertilization, they share a fundamental result:

13 Each cycle of chromosome halving and doubling contributes to genetic variation among offspring.

13 A closer look at meiosis will reveal the sources of this variation.

13 Many of the steps of meiosis closely resemble corresponding steps in mitosis.

13 Meiosis, like mitosis, is preceded by the replication of chromosomes.

13 However, this single replication is followed by two consecutive cell divisions, called meiosis I and meiosis II.

13 These divisions result in four daughter cells (rather than the two daughter cells of mitosis), each with only half as many chromosomes as the parent cell.

13 Study the overview of meiosis in FIGURE 13.6, and be sure you understand the difference between homologous chromosomes and sister chromatids.

13 The two chromosomes of a homologous pair are individual chromosomes that were inherited from different parents.

13 Homologues appear alike in the microscope, but they have different versions of genes at some of their corresponding loci (for example, a gene for freckles on one chromosome and a gene for the absence of freckles at the same locus on the homologue).

13 The second meiotic division, meiosis II, separates sister chromatids and is virtually identical in mechanism to mitosis.

13 However, since the chromosomes do not replicate between meiosis I and meiosis II, the final outcome of meiosis is a halving of the number of chromosomes per cell--a reduction from two haploid sets to one haploid set in each cell.

13 In species that reproduce sexually, the behavior of chromosomes during meiosis and fertilization is responsible for most of the variation that arises each generation.

13 One way sexual reproduction generates genetic variation is shown in FIGURE 13.9, which features meiosis of a diploid cell with two homologous pairs of chromosomes.

13 The red and blue colors distinguishing the maternal and paternal chromosomes of each homologous pair allow us to track individual chromosomes as meiosis proceeds and they are packaged in gametes.

13 At metaphase I, the homologous pairs of chromosomes, each consisting of one maternal and one paternal chromosome, are situated on the metaphase plate.

13 The orientations of the homologous pairs relative to the poles of the cell are random; there are two alternative possibilities for each pair.

13 Thus, there is a fifty-fifty chance that a particular daughter cell of meiosis I will get the maternal chromosome of a certain homologous pair and a fifty-fifty chance that it will receive the paternal chromosome.

13 Fig 13-9. The results of alternative arrangements of two homologous chromosome pairs on the metaphase plate in meiosis I.

13 In this figure we consider the consequences of meiosis in a hypothetical organism with a diploid chromosome number of 4 (2n = 4).

13 The parental origins of the chromosomes are designated with different colors, blue for chromosomes inherited from one parent, red for chromosomes from the other parent.

13 The positioning of each homologous pair of chromosomes at metaphase I is a matter of chance and determines which chromosomes will be packaged together in the haploid daughter cells.

13 Because each homologous pair of chromosomes is positioned independently of the other pairs at metaphase I--its orientation is as random as the flip of a coin--the first meiotic division results in independent assortment of maternal and paternal chromosomes into daughter cells.

13 Each gamete represents one outcome of all possible combinations of maternal and paternal chromosomes.

13 The number of combinations possible for gametes formed by meiosis starting with two homologous pairs of chromosomes (2n = 4, n = 2) is four, as shown in FIGURE 13.9. (Only two of the four combinations of gametes shown in the figure would result from meiosis of a single diploid cell, but starting with a large number of diploid cells, gametes of all four types would be produced in approximately equal numbers.)

13 In the case of n = 3, eight combinations of chromosomes are possible for gametes.

13 More generally, the number of combinations possible when chromosomes assort independently into gametes during meiosis is 2n, where n is the haploid number of the organism.

13 In the case of humans, the haploid number (n) in the formula is 23.

13 Thus, the number of possible combinations of maternal and paternal chromosomes in the resulting gametes is 223, or about 8 million.

13 The variety of gametes is analogous to the different combinations of heads and tails possible for the simultaneous tossing of 23 coins.

13 Thus, each gamete that a human produces contains one of roughly 8 million possible assortments of chromosomes inherited from that individual’s mother and father.

13 As a consequence of the independent assortment of chromosomes during meiosis, each of us produces a collection of gametes differing greatly in their combinations of the chromosomes we inherited from our two parents.

13 But from what you have learned so far, it would seem that each individual chromosome in a gamete would be exclusively maternal or paternal in origin; that is, it would consist of DNA derived from our mother or father, but not from both.

13 The process called crossing over produces recombinant chromosomes, which combine genes inherited from our two parents (FIGURE 13.10).

13 Following these chromosomes through meiosis, we can see that crossing over gives rise to recombinant chromosomes, individual chromosomes that have some combination of DNA originally derived from two different parents.

13 Recent research has revealed that crossing over begins very early in prophase I, as homologous chromosomes pair loosely along their lengths and before the synaptonemal complex forms between them.

13 The pairing is precise, the homologues aligning with each other gene by gene.

13 In crossing over, homologous portions of two nonsister chromatids trade places. (For humans, an average of two or three such crossover events occur per chromosome pair.)

13 After the synaptonemal complex disappears, the locations where these genetic exchanges have occurred are visible as chiasmata.

13 At metaphase II, chromosomes that contain one or more recombinant chromatids can be oriented in two alternative, nonequivalent ways with respect to other chromosomes, because their sister chromatids are no longer identical twins.

13 The independent assortment of these non-identical sister chromatids during meiosis II increases still more the number of genetic types of gametes that can result from meiosis.

13 You will learn more about crossing over in Chapter 15.

13 The important point for now is that crossing over, by combining DNA inherited from two parents into a single chromosome, is an important source of genetic variation in sexual life cycles.

13 The random nature of fertilization adds to the genetic variation arising from meiosis.

13 Consider a zygote resulting from a mating between a woman and a man.

13 A human ovum, representing one of approximately 8 million possible chromosome combinations, is fertilized by a single sperm cell, which represents one of 8 million different possibilities.

13 Thus, even without considering crossing over, any two parents will produce a zygote with any of about 64 trillion (8 million X 8 million) diploid combinations. (If you calculate 223 X 223 exactly, you will find that the total is actually over 70 trillion.)

13 Adding in the variation brought about by crossing over, the number of possibilities is truly astronomical.

13 No wonder brothers and sisters can be so different.

13 You really are unique.

13 So far, we have seen that there are three sources of genetic variability in a sexually reproducing population of organisms

13 Crossing over between homologous chromosomes during prophase of meiosis I.

13 Random fertilization of an ovum by a sperm.

13 All three mechanisms reshuffle the various genes carried by the individual members of a population.

13 However, as you will learn in subsequent chapters, mutations are what ultimately create a population’s diversity of genes.

13 Having considered how sexual reproduction contributes to genetic variation in a population, we can relate these concepts to evolution, biology’s core theme.

13 Darwin recognized the importance of genetic variation in the evolutionary mechanism he called natural selection.

13 Recall from Chapter 1 that a population evolves through the differential reproductive success of its variant members.

13 On average, those individuals best suited to the local environment leave the most offspring, transmitting their genes in the process.

13 This natural selection results in adaptation, the accumulation of the genetic variations favored by the environment.

13 As the environment changes or a population moves, the population may survive if, in each generation, at least some of its members can cope effectively with the new conditions.

13 Different genetic variations may work better than those that prevailed in the old time or place.

13 Sex and mutations are the two sources of this variation, and we have considered the sexual contribution in this chapter.

13 Although Darwin realized that heritable variation is what makes evolution possible, he could not explain why offspring resemble--but are not identical to--their parents.

13 Ironically, Gregor Mendel, a contemporary of Darwin, published a theory of inheritance that helps explain genetic variation, but his discoveries had no impact on biologists until 1900, more than 15 years after Darwin (1809-1882) and Mendel (1822-1884) had died.

13 In the next chapter, you will learn how Mendel discovered the basic rules governing the inheritance of specific traits.

16 In a second paper that followed their announcement of the double helix, Watson and Crick stated their hypothesis for how DNA replicates:

16 Now our model for deoxyribonucleic acid is, in effect, a pair of templates, each of which is complementary to the other.

16 We imagine that prior to duplication the hydrogen bonds are broken, and the two chains unwind and separate.

16 Each chain then acts as a template for the formation onto itself of a new companion chain, so that eventually we shall have two pairs of chains, where we only had one before.

16 Moreover, the sequence of the pairs of bases will have been duplicated exactly.*

16 FIGURE 16.7 illustrates Watson and Crick’s basic idea.

16 To make it easier to follow, the diagram shows only a short section of double helix, in untwisted form.

16 Notice that if you cover one of the two DNA strands of FIGURE 16.7a, you can still determine its linear sequence of bases by referring to the unmasked strand and applying the base-pairing rules.

16 The two strands are complementary; each stores the information necessary to reconstruct the other.

16 When a cell copies a DNA molecule, each strand serves as a template (mold) for ordering nucleotides into a new complementary strand.

16 The nucleotides are linked to form the new strands.

16 Where there was one double-stranded DNA molecule at the beginning of the process, there are now two, each an exact replica of the "parent" molecule.

16 The copying mechanism is analogous to using a photographic negative to make a positive image, which can in turn be used to make another negative, and so on. (See FIGURE 5.30 for a helical version of FIGURE 16.7.)

16 Fig 16-7. A model for DNA replication: the basic concept.

16 In this simplification, a short segment of DNA has been untwisted into a structure that resembles a ladder.

16 The rails of the ladder are the sugar-phosphate backbones of the two DNA strands; the rungs are the pairs of nitrogenous bases.

16 This model of gene replication remained untested for several years following publication of the DNA structure.

16 The requisite experiments were simple in concept but difficult to perform.

16 Watson and Crick’s model predicts that when a double helix replicates, each of the two daughter molecules will have one old strand, derived from the parent molecule, and one newly made strand.

16 This semiconservative model can be distinguished from a conservative model of replication, in which the parent molecule somehow emerges from the replication process intact (that is, it is conserved).

16 In yet a third model, called the dispersive model, all four strands of DNA following replication have a mixture of old and new DNA (FIGURE 16.8, p. 294).

16 Although mechanisms for conservative or dispersive DNA replication are not easy to devise, these models remained possibilities until they could be ruled out.

16 Finally, in the late 1950s, Matthew Meselson and Franklin Stahl devised experiments that tested the three hypotheses.

16 Meselson and Stahl cultured E. coli bacteria for several generations on a medium containing a heavy isotope of nitrogen, 15N.

16 The bacteria incorporated the heavy nitrogen into their nucleotides and then into their DNA.

16 The scientists then transferred the bacteria to a medium containing 14N, the lighter, more common isotope of nitrogen.

16 Any new DNA that the bacteria synthesized would be lighter than the "old" DNA made in the 15N medium

16 Meselson and Stahl could distinguish DNA of different densities by centrifuging DNA extracted from the bacteria.

16 The centrifuge tubes in this drawing represent the results predicted by each of the three models in FIGURE 16.8.

16 The first replication in the 14N medium produced a band of hybrid (15N-14N) DNA.

16 This result eliminated the conservative model.

16 A second replication produced both light and hybrid DNA, a result that eliminated the dispersive model and supported the semiconservative model.

16 The basic principle of DNA replication is elegantly simple.

16 However, the actual process involves complex biochemical gymnastics, as we will now see.

16 A large team of enzymes and other proteins carries out DNA replication\

16 The bacterium E. coli has a single chromosome of about 5 million base pairs.

16 In a favorable environment, an E. coli cell can copy all this DNA and divide to form two genetically identical daughter cells in less than an hour.

16 Each of your cells has 46 DNA molecules in its nucleus, one giant molecule per chromosome.

16 In all, that represents about 6 billion base pairs, or over a thousand times more DNA than is found in a bacterial cell.

16 If we were to print the one-letter symbols for these bases (A, G, C, and T) the size of the letters you are now reading, the 6 billion bases of a single human cell would fill about 900 books as thick as this text.

16 Yet it takes a cell just a few hours to copy all this DNA.

16 This replication of an enormous amount of genetic information is achieved with very few errors--only one per billion nucleotides.

16 The copying of DNA is remarkable in its speed and accuracy.

16 More than a dozen enzymes and other proteins participate in DNA replication.

16 Much more is known about how this "replication machine" works in bacteria than in eukaryotes.

16 However, most of the process seems to be fundamentally similar for prokaryotes and eukaryotes.

16 In this section, we take a closer look at the basic steps.

16 The replication of a DNA molecule begins at special sites called origins of replication.

16 The bacterial chromosome, which is circular, has a single origin, a stretch of DNA having a specific sequence of nucleotides.

16 Proteins that initiate DNA replication recognize this sequence and attach to the DNA, separating the two strands and opening up a replication "bubble."

16 Replication of DNA then proceeds in both directions, until the entire molecule is copied (see FIGURE 18.11).

16 In contrast to the bacterial chromosome, a eukaryotic chromosome may have hundreds or even thousands of replication origins.

16 Multiple replication bubbles form and eventually fuse, thus speeding up the copying of the very long DNA molecules (FIGURE 16.10).

16 As in bacteria, DNA replication proceeds in both directions from each origin.

16 At each end of a replication bubble is a replication fork, a Y-shaped region where the new strands of DNA are elongating.

16 Elongation of new DNA at a replication fork is catalyzed by enzymes called DNA polymerases.

16 As nucleotides align with complementary bases along a template strand of DNA, they are added by polymerase, one by one, to the growing end of the new DNA strand.

16 The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells.

16 What is the source of energy that drives the polymerization of nucleotides to form new DNA strands?

16 The nucleotides that serve as substrates for DNA polymerase are actually nucleo side triphosphates, which are nucleotides with three phosphate groups (FIGURE 16.11, p. 296).

16 You have already encountered such a molecule--ATP.

16 The only difference between the ATP of energy metabolism and the nucleoside triphosphate that supplies adenine to DNA is the sugar component, which is deoxyribose in the building block of DNA, but ribose in ATP. (As you might guess, ribose-containing ATP is a substrate for RNA synthesis.)

16 Like ATP, the triphosphate monomers used for DNA synthesis are chemically reactive, partly because their triphosphate tails have an unstable cluster of negative charge.

16 As each monomer joins the growing end of a DNA strand, it loses two phosphate groups as a molecule of pyrophosphate P - Pi).

16 Subsequent hydrolysis of the pyrophosphate to two molecules of inorganic phosphate (Pi) is the exergonic reaction that drives the polymerization reaction.

16 Fig 16-11. Incorporation of a nucleotide into a DNA strand.

16 When a nucleoside triphosphate links to the sugar-phosphate backbone of a growing DNA strand, it loses two of its phosphates as a pyrophosphate molecule.

16 The enzyme catalyzing the reaction is a DNA polymerase, and hydrolysis of the bonds between the phosphate groups of the pyrophosphate provides the energy for the reaction.

16 There is more to the scenario of DNA synthesis at the replication fork.

16 Until now, we have ignored an important feature of the double helix:

16 The two DNA strands are antiparallel; that is, their sugar-phosphate backbones run in opposite directions.

16 In FIGURE 16.12, the five carbons of one deoxyribose sugar of each DNA strand are numbered from 1' to 5'. (The prime sign is used to distinguish the carbon atoms of the sugar from the carbon and nitrogen atoms of the nitrogenous bases.)

16 Notice in FIGURE 16.12 that a nucleotide’s phosphate group is attached to the 5' carbon of deoxyribose.

16 Notice also that the phosphate group of one nucleotide is joined to the 3' carbon of the adjacent nucleotide.

16 The result is a DNA strand of distinct polarity.

16 At one end, denoted the 3' end, a hydroxyl group is attached to the 3' carbon of the terminal deoxyribose.

16 At the opposite end, the 5' end, the sugar-phosphate backbone terminates with the phosphate group attached to the 5' carbon of the last nucleotide.

16 In the double helix, the two sugar-phosphate backbones are essentially upside down (antiparallel) relative to each other.

16 Fig 16-12. The two strands of DNA are antiparallel.

16 The numbers assigned to the carbon atoms of the deoxyribose units are shown for two of them.

16 How does the antiparallel structure of the double helix affect replication?

16 DNA polymerases add nucleotides only to the free 3' end of a growing DNA strand, never to the 5' end.

16 Thus, a new DNA strand can elongate only in the 5' 3' direction.

16 With this in mind, let’s examine a replication fork (FIGURE 16.13).

16 Along one template strand, DNA polymerase can synthesize a continuous complementary strand by elongating the new DNA in the mandatory 5' 3' direction.

16 The polymerase simply nestles in the replication fork on that template strand and continuously adds nucleotides to a complementary strand as the fork progresses.

16 The DNA strand made by this mechanism is called the leading strand.

16 To elongate the other new strand of DNA, polymerase must work along the other template strand in the direction away from the replication fork.

16 The DNA synthesized in this direction is called the lagging strand.

16 The process is analogous to a sewing method called backstitching.

16 As a replication bubble opens, a polymerase molecule can work its way away from a replication fork and synthesize a short segment of DNA.

16 As the bubble grows, another short segment of the lagging strand can be made in a similar way.

16 In contrast to the leading strand, which elongates continuously, the lagging strand is first synthesized as a series of segments.

16 These pieces are called Okazaki fragments, after the Japanese scientist who discovered them.

16 The fragments are about 100 to 200 nucleotides long in eukaryotes.

16 Another enzyme, DNA ligase, joins (ligates) the sugar-phosphate backbones of the Okazaki fragments to create a single DNA strand.

16 There is another important restriction for DNA polymerases.

16 None of these enzymes can actually initiate synthesis of a polynucleotide; they can only add nucleotides to the end of an already existing chain that is base-paired with the template strand (see FIGURE 16.11).

16 In the replication of cellular DNA, the start of a new chain, its primer, is not DNA, but a short stretch of RNA, the other class of nucleic acid.

16 An enzyme called primase joins RNA nucleotides to make the primer, which is about 10 nucleotides long in eukaryotes

16 Like all RNA-synthesizing enzymes, primase can start an RNA chain from scratch

16 Another DNA polymerase later replaces the RNA nucleotides of the primers with DNA versions.

16 Only one primer is required for a DNA polymerase to begin synthesizing the leading strand of new DNA.

16 For the lagging strand, each Okazaki fragment must be primed; the primers are converted to DNA before ligase joins the fragments together.

16 Fig 16-14. Priming DNA synthesis with RNA.

16 DNA polymerase cannot initiate a polynucleotide strand; it can only add to the 3' end of an already-started strand.

16 The primer is a short segment of RNA synthesized by the enzyme primase.

16 Each primer is eventually replaced by DNA.

16 You have learned about three kinds of proteins that function in DNA synthesis: DNA polymerase, ligase, and primase.

16 Other kinds of proteins also participate; two of these are helicase and single-strand binding protein.

16 A helicase is an enzyme that untwists the double helix at the replication fork, separating the two old strands.

16 Molecules of single-strand binding protein then line up along the unpaired DNA strands, holding them apart while they serve as templates for the synthesis of new complementary strands.

16 Fig 16-16. A summary of DNA replication.

16 The detailed diagram shows one replication fork, but as indicated in the overview diagram, replication usually occurs simultaneously at two forks, one at either end of a replication bubble.

16 Notice in the overview diagram that a leading strand is initiated by an RNA primer (magenta), as is each Okazaki fragment in a lagging strand.

16 Viewing each daughter strand in its entirety, you can see that half of it is made continuously as a leading strand, while the other half (on the other side of the origin) is synthesized in fragments as a lagging strand.

16 It is traditional--and convenient--to represent DNA polymerase molecules as locomotives moving along a DNA "railroad track," but such a model is inaccurate in two important ways.

16 First, the various proteins that participate in DNA replication actually form a single large complex, a DNA replication "machine."

16 Second, this machine is probably stationary during the replication process.

16 In eukaryotic cells, the multiple copies of the machine, perhaps grouped into "factories," may anchor to the nuclear matrix, a framework of fibers extending through the interior of the nucleus.

16 Recent studies support a model in which DNA polymerase molecules "reel in" the parental DNA and "extrude" newly made daughter DNA molecules.

16 Enzymes proofread DNA during its replication and repair damage in existing DNA

16 We cannot attribute the accuracy of DNA replication solely to the specificity of base pairing.

16 Although errors in the completed DNA molecule amount to only one in a billion nucleotides, initial pairing errors between incoming nucleotides and those in the template strand are 100,000 times more common--an error rate of one in 10,000 base pairs.

16 During DNA replication, DNA polymerase itself proofreads each nucleotide against its template as soon as it is added to the growing strand.

16 Upon finding an incorrectly paired nucleotide, the polymerase removes the nucleotide and then resumes synthesis.

16 This action resembles correcting a word-processing error by using the "delete" key and then entering the correction.

16 Mismatched nucleotides sometimes evade proofreading by DNA polymerase or arise after DNA synthesis is completed--by damage to a nucleotide base, for instance.

16 In mismatch repair, cells use special enzymes to fix incorrectly paired nucleotides.

16 Researchers spotlighted the importance of such proteins when they found that a hereditary defect in one of them is associated with a form of colon cancer.

16 Apparently, this defect allows cancer-causing errors to accumulate in the DNA.

16 Maintenance of the genetic information encoded in DNA requires frequent repair of various kinds of damage to existing DNA.

16 Fortunately, changes in DNA are usually corrected before they become self-perpetuating mutations.

16 Each cell continuously monitors and repairs its genetic material.

16 Because repair of damaged DNA is so important to the survival of an organism, it is no surprise that many different DNA repair enzymes have evolved.

16 Almost 100 are known in E. coli, and 130 have been identified so far in humans.

16 Most mechanisms for repairing DNA damage take advantage of the base-paired structure of DNA.

16 Usually, a segment of the strand containing the damage is cut out (excised) by a DNA-cutting enzyme--a nuclease--and the resulting gap is filled in with nucleotides properly paired with the nucleotides in the undamaged strand.

16 DNA repair of this type is called nucleotide excision repair (FIGURE 16.17).

16 Fig 16-17. Nucleotide excision repair of DNA damage.

16 A team of enzymes detects and repairs damaged DNA.

16 This figure shows DNA containing a thymine dimer, a type of damage often caused by ultra-violet radiation.

16 Repair enzymes can excise the damaged region from the DNA and replace it with a normal DNA segment

16 One function of the DNA repair enzymes in our skin cells is to repair genetic damage caused by the ultraviolet rays of sunlight.

16 One type of damage, the type shown in FIGURE 16.17, is the covalent linking of thymine bases that are adjacent on a DNA strand.

16 Such thymine dimers cause the DNA to buckle and interfere with DNA replication.

16 The importance of repairing this kind of damage is underscored by the disorder xeroderma pigmentosum, which in most cases is caused by an inherited defect in a nucleotide excision repair enzyme.

16 Individuals with this disorder are hypersensitive to sunlight; mutations in their skin cells caused by ultraviolet light are left uncorrected and cause skin cancer.

16 Most DNA repair processes involve DNA polymerases, but these enzymes are helpless to fix a "defect" that results from their own limitations.

16 For linear DNA, such as the DNA of eukaryotic chromosomes, the fact that a DNA polymerase can only add nucleotides to the 3' end of a preexisting polynucleotide leads to a potential problem.

16 Clearly, if this trend continued over generations, we would not be here today!

16 Fig 16-18. The end-replication problem.

16 When a linear DNA molecule replicates, a gap is left at the 5' end of each new strand (light blue) because DNA polymerase can only add nucleotides to a 3' end.

16 As a result, with each round of replication, the DNA molecules get slightly shorter.

16 For simplicity we show only one end of a linear DNA molecule.

16 Prokaryotes avoid this problem by having circular DNA molecules (which have no ends), but what about eukaryotes?

16 Eukaryotic chromosomal DNA molecules have special nucleotide sequences called telomeres at their ends (FIGURE 16.19).

16 Telomeres do not contain genes; instead, the DNA consists of multiple repetitions of one short nucleotide sequence.

16 The repeated unit in human telomeres, which is typical, is the six-nucleotide sequence TTAGGG.

16 The number of repetitions in a telomere varies between about 100 and 1,000.

16 Telomeric DNA protects the organism’s genes from being eroded through successive rounds of DNA replication.

16 In addition, telomeric DNA and special proteins associated with it somehow prevent the ends from activating the cell’s systems for monitoring DNA damage.

16 The end of a DNA molecule that is "seen" as a double-strand break may otherwise trigger signal-transduction pathways leading to cell cycle arrest or cell death.

16 Fig 16-19. Telomeres and telomerase.

16 Eukaryotes deal with the end-replication issue by having expendable, noncoding sequences called telomeres at the ends of their DNA and the enzyme telomerase in some of their cells.

16 In the long term, over the course of generations, eukaryotic organisms need a way of restoring their shortened telomeres.

16 This is provided by telomerase, a special enzyme that catalyzes the lengthening of telomeres.

16 But how does telomerase synthesize DNA where the DNA template has been lost?

16 Telomerase is unusual in having a short molecule of RNA along with its protein.

16 The RNA contains a nucleotide sequence that serves as the template for new telomere segments at the 3' end of the telo-mere.

16 FIGURE 16.19 shows how telomerase and DNA polymerase work together to lengthen telomeres.

16 Telomerase is not present in most cells of multicellular organisms like ourselves, and the DNA of dividing somatic cells does tend to be shorter in older individuals and in cultured cells that have divided many times.

16 Thus, it is possible that telomeres are a limiting factor in the life span of certain tissues and even the organism as a whole.

16 In any case, telomerase is present in germ-line cells, those that give rise to gametes.

16 The enzyme produces long telomeres in these cells and hence in the newborn.

16 Intriguingly, researchers have also found telomerase in somatic cells that are cancerous.

16 Cells from large tumors often have unusually short telomeres, as one would expect for cells that have undergone many cell divisions.

16 Progressive shortening would presumably lead eventually to self-destruction of the cancer unless telomerase became available to stabilize telomere length.

16 This is exactly what seems to happen in cancer cells and also in immortal strains of cultured cells (see Chapter 12).

16 If telomerase is indeed an important factor in many cancers, it may provide a useful target for both cancer diagnosis and chemotherapy.

16 However, it is not enough that genes be copied and transmitted; they must also be expressed.

16 How do genes manifest themselves in phenotypic characters such as eye color?

16 In the next chapter, we will examine the molecular basis of gene expression--how the cell translates genetic information encoded in DNA.

17 In 1909, British physician Archibald Garrod was the first to suggest that genes dictate phenotypes through enzymes that catalyze specific chemical reactions in the cell.

17 Garrod postulated that the symptoms of an inherited disease reflect a person’s inability to make a particular enzyme.

17 He referred to such diseases as "inborn errors of metabolism."

17 Garrod gave as one example the hereditary condition called alkaptonuria, in which the urine is black because it contains the chemical alkapton, which darkens upon exposure to air.

17 Garrod reasoned that normal individuals have an enzyme that breaks down alkapton, whereas alkaptonuric individuals have inherited an inability to make the enzyme that metabolizes alkapton.

17 How Genes Control Metabolism: One Gene-One Enzyme

17 Garrod’s idea was ahead of its time, but research conducted several decades later supported his hypothesis that a gene dictates the production of a specific enzyme.

17 Biochemists accumulated much evidence that cells synthesize and degrade most organic molecules via metabolic pathways, in which each chemical reaction in a sequence is catalyzed by a specific enzyme.

17 Such metabolic pathways lead, for instance, to the synthesis of the pigments that give fruit flies (Drosophila) their eye color (see FIGURE 15.2).

17 In the 1930s, George Beadle and Boris Ephrussi speculated that each of the various mutations affecting eye color in Drosophila blocks pigment synthesis at a specific step by preventing production of the enzyme that catalyzes that step.

17 However, neither the chemical reactions nor the enzymes that catalyze them were known at the time.

17 A breakthrough in demonstrating the relationship between genes and enzymes came a few years later, when Beadle and Edward Tatum began working with a bread mold, Neurospora crassa.

17 They treated Neurospora with X-rays and then looked among the survivors for mutants that differed from the wild-type mold in their nutritional needs.

17 Wild-type Neurospora has modest food requirements.

17 It can survive in the laboratory on agar (a moist support medium) mixed only with inorganic salts, glucose, and the vitamin biotin.

17 From this minimal medium, the mold uses its metabolic pathways to produce all the other molecules it needs.

17 Beadle and Tatum identified mutants that could not survive on minimal medium, apparently because they were unable to synthesize certain essential molecules from the minimal ingredients.

17 However, most such nutritional mutants can survive on a complete growth medium, minimal medium supplemented with all 20 amino acids and a few other nutrients.

17 To characterize the metabolic defect in each nutritional mutant, Beadle and Tatum took samples from the mutant growing on complete medium and distributed them to a number of different vials.

17 For example, if the only supplemented vial that supported growth of the mutant was the one fortified with the amino acid arginine, the researchers could conclude that the mutant was defective in the biochemical pathway that wild-type cells use to synthesize arginine.

17 Beadle and Tatum went on to pin down each mutant’s defect more specifically.

17 Their work with arginine-requiring mutants was especially instructive.

17 Using genetic crosses, they determined that their mutants fell into three classes, each mutated in a different gene.

17 The researchers then showed that they could distinguish among the classes of mutants nutritionally by additional tests of their growth requirements (FIGURE 17.1).

17 In the synthetic pathway leading to arginine, they suspected, a precursor nutrient is converted to ornithine, which is converted to citrulline, which is converted to arginine.

17 When they tested their arginine mutants for growth on ornithine and citrulline, they found that one class could grow on either compound (or arginine), the second class only on citrulline (or arginine), and the third on neither--it absolutely required arginine.

17 The three classes of mutants, the researchers reasoned, must be blocked at different steps in the pathway that synthesizes arginine, with each mutant class lacking the enzyme that catalyzes the blocked step.

17 Fig 17-1. Beadle and Tatum’s evidence for the one gene-one enzyme hypothesis.

17 In this experiment, the researchers studied three classes of mutants of the mold Neurospora crassa, all defective in synthesizing the amino acid arginine.

17 The wild-type strain requires only a minimal nutritional medium for growth; it makes arginine by using a multistep pathway in which ornithine and citrulline are intermediates.

17 The different types of mutants had different growth requirements.

17 Beadle and Tatum concluded that each gene mutated must normally dictate the production of one enzyme: the one gene--one enzyme hypothesis.

17 Because each mutant was defective in a single gene, Beadle and Tatum’s results provided strong support for the one gene-one enzyme hypothesis, as they dubbed it, which states that the function of a gene is to dictate the production of a specific enzyme.

17 The researchers also showed how a combination of genetics and biochemistry could be used to work out the steps in a metabolic pathway.

17 Further support for the one gene-one enzyme hypothesis came with biochemical experiments that identified the specific enzymes lacking in the mutants.

17 As researchers learned more about proteins, they made minor revisions in the one gene-one enzyme hypothesis.

17 Not all proteins are enzymes.

17 Keratin, the structural protein of animal hair, and the hormone insulin are two examples of nonenzyme proteins.

17 Because proteins that are not enzymes are nevertheless gene products, molecular biologists began to think in terms of one gene-one protein.

17 However, many proteins are constructed from two or more different polypeptide chains, and each polypeptide is specified by its own gene.

17 For example, hemoglobin, the oxygen-transporting protein of vertebrate red blood cells, is built from two kinds of polypeptides, and thus two genes code for this protein (see FIGURE 5.23b).

17 We can therefore restate Beadle and Tatum’s idea as the one gene-one polypeptide hypothesis.

17 Note, however, that it is common to refer to proteins, rather than polypeptides, as the gene products, a practice you will encounter in this book.

17 Transcription and translation are the two main processes linking gene to protein: an overview

17 Genes provide the instructions for making specific proteins.

17 But a gene does not build a protein directly.

17 The bridge between DNA and protein synthesis is RNA.

17 You learned in Chapter 5 that RNA is chemically similar to DNA, except that it contains ribose instead of deoxyribose as its sugar and has the nitrogenous base uracil rather than thymine (see FIGURE 5.29).

17 Thus, each nucleotide along a DNA strand has deoxyribose as its sugar and A, G, C, or T as its base; each nucleotide along an RNA strand has ribose as its sugar and A, G, C, or U as its base.

17 An RNA molecule almost always consists of a single strand.

17 It is customary to describe the flow of information from gene to protein in linguistic terms because both nucleic acids and proteins are polymers with specific sequences of monomers that convey information, much as specific sequences of letters communicate information in a language like English.

17 Genes are typically hundreds or thousands of nucleotides long, each gene having a specific sequence of bases.

17 Each polypeptide of a protein also has monomers arranged in a particular linear order (the protein’s primary structure), but its monomers are the 20 amino acids.

17 Thus, nucleic acids and proteins contain information written in two different chemical languages.

17 To get from DNA, written in one language, to protein, written in the other, requires two major stages, transcription and translation

17 Transcription is the synthesis of RNA under the direction of DNA.

17 Both nucleic acids use the same language, and the information is simply transcribed, or copied, from one molecule to the other.

17 Just as a DNA strand provides a template for the synthesis of a new complementary strand during DNA replication, it provides a template for assembling a sequence of RNA nucleotides.

17 The resulting RNA molecule is a faithful transcript of the gene’s protein-building instructions.

17 This type of RNA molecule is called messenger RNA (mRNA), because it carries a genetic message from the DNA to the protein-synthesizing machinery of the cell (FIGURE 17.2a).

17 Transcription is the general term for the synthesis of any kind of RNA on a DNA template.

17 Later in this chapter, you will learn about other types of RNA produced by transcription.

17 Fig 17-2. Overview: the roles of transcription and translation in the flow of genetic information.

17 In a cell, inherited information flows from DNA to RNA to protein.

17 The two main stages of information flow are transcription and translation.

17 In translation, the information encoded in mRNA determines the order of amino acids that are joined to form a specific polypeptide.

17 The sites of translation are ribosomes.

17 A miniature version of part b (or sometimes part a) accompanies several figures later in the Chapter as an orientation diagram to help you see where a particular figure fits into the overall scheme.

17 Translation is the actual synthesis of a polypeptide, which occurs under the direction of mRNA.

17 During this stage, there is a change in language:

17 The cell must translate the base sequence of an mRNA molecule into the amino acid sequence of a polypeptide.

17 The sites of translation are ribosomes, complex particles that facilitate the orderly linking of amino acids into polypeptide chains.

17 Although the basic mechanics of transcription and translation are similar for prokaryotes and eukaryotes, there is an important difference in the flow of genetic information within the cells.

17 Because bacteria lack nuclei, their DNA is not segregated from ribosomes and the other protein-synthesizing equipment.

17 Transcription and translation are coupled, with ribosomes attaching to the leading end of an mRNA molecule while transcription is still in progress (see FIGURE 17.22).

17 In a eukaryotic cell, by contrast, the nuclear envelope separates transcription from translation in space and time (FIGURE 17.2b)

17 Transcription occurs in the nucleus, and mRNA is dispatched to the cytoplasm, where translation occurs.

17 But before they can leave the nucleus, eukaryotic RNA transcripts are modified in various ways to produce the final, functional mRNA.

17 Thus, in a two-step process, the transcription of a eukaryotic gene results in pre-mRNA, and RNA processing yields the finished mRNA.

17 A more general term for an initial RNA transcript is primary transcript.

17 Let’s summarize the main point of our overview of protein synthesis:

17 Genes program protein synthesis via genetic messages in the form of messenger RNA.

17 Put another way, cells are governed by a molecular chain of command: DNA RNA protein.

17 The next section discusses how the instructions for assembling amino acids into a specific order are encoded in nucleic acids.

17 In the genetic code, nucleotide triplets specify amino acids

17 When biologists began to suspect that the instructions for protein synthesis were encoded in DNA, they recognized a problem:

17 There are only four nucleotides to specify 20 amino acids.

17 Thus, the genetic code cannot be a language like Chinese, where each written symbol corresponds to a single word.

17 If each nucleotide base were translated into an amino acid, only 4 of the 20 amino acids could be specified.

17 \\Would a language of two-letter code words suffice?

17 The base sequence AG, for example, could specify one amino acid, and GT could specify another.

17 Since there are four bases, this would give us 16 (that is, 42) possible arrangements--still not enough to code for all 20 amino acids.

17 Triplets of nucleotide bases are the smallest units of uniform length that can code for all the amino acids.

17 If each arrangement of three consecutive bases specifies an amino acid, there can be 64 (that is, 43) possible code words--more than enough to specify all the amino acids.

17 Experiments have verified that the flow of information from gene to protein is based on a triplet code:

17 The genetic instructions for a polypeptide chain are written in the DNA as a series of three-nucleotide words.

17 For example, the base triplet AGT at a particular position along a DNA strand results in the placement of the amino acid serine at the corresponding position of the polypeptide to be produced.

17 As you know, a cell does not directly translate a gene into amino acids.

17 The intermediate step is transcription, during which the gene determines the sequence of base triplets along the length of an mRNA molecule.

17 For each gene, only one of the two DNA strands is transcribed (FIGURE 17.3).

17 This strand is called the template strand, because it provides the template for ordering the sequence of nucleotides in an RNA transcript. ``

17 A given DNA strand can be the template strand in some regions of a DNA molecule, while in other regions along the double helix it is the complementary strand that functions as the template for RNA synthesis.

17 Fig 17-3. The triplet code.

17 For each gene, one DNA strand functions as a template for transcription--the synthesis of a complementary mRNA molecule.

17 The base-pairing rules for DNA synthesis also guide transcription, but uracil (U) takes the place of thymine (T) in RNA.

17 During translation, the mRNA is read as a sequence of base triplets, called codons.

17 Each codon specifies an amino acid to be added to the growing polypeptide chain.

17 The mRNA is read in the 5' 3' direction.

17 An mRNA molecule is complementary rather than identical to its DNA template because RNA bases are assembled on the template according to base-pairing rules.

17 The pairs are similar to those that form during DNA replication, except that U, the RNA substitute for T, pairs with A.

17 Thus, when a DNA strand is transcribed, the base triplet ACC in DNA provides a template for UGG in the mRNA molecule.

17 The mRNA base triplets are called codons.

17 For example, UGG is the codon for the amino acid tryptophan (abbreviated Trp).

17 The term codon is also sometimes used for the complementary DNA base triplet.

17 For example, the DNA codon corresponding to the RNA codon UGG is ACC.

17 During translation, the sequence of codons along an mRNA molecule is decoded, or translated, into a sequence of amino acids making up a polypeptide chain.

17 The codons are read in the 5' 3' direction along the mRNA.

17 To review what is meant by the 5' and 3' ends of a nucleic acid chain, see FIGURE 16.12

17 Each codon specifies which one of the 20 amino acids will be incorporated at the corresponding position along a polypeptide.

17 Because codons are base triplets, the number of nucleotides making up a genetic message must be three times the number of amino acids making up the protein product.

17 For example, it takes 300 nucleotides along an RNA strand to code for a polypeptide that is 100 amino acids long.

17 Cracking the Genetic Code

17 Molecular biologists cracked the code of life in the early 1960s, when a series of elegant experiments disclosed the amino acid translations of each of the RNA codons.

17 The first codon was deciphered in 1961 by Marshall Nirenberg, of the National Institutes of Health, and his colleagues.

17 Soon, the amino acids specified by the codons AAA, GGG, and CCC were also determined.

17 Although more elaborate techniques were required to decode mixed triplets such as AUA and CGA, all 64 codons were deciphered by the mid-1960s.

17 As FIGURE 17.4 shows, 61 of the 64 triplets code for amino acids.

17 Notice that the codon AUG has a dual function:

17 It not only codes for the amino acid methionine (Met), but also functions as a "start" signal, or initiation codon.

17 Genetic messages begin with the mRNA codon AUG, which signals the protein-synthesizing machinery to begin translating the mRNA at that location.

17 Because AUG also stands for methionine, polypeptide chains begin with methionine when they are synthesized.

17 However, an enzyme may subsequently remove this starter amino acid from a chain.

17 The remaining three codons do not designate amino acids.

17 Instead, they are "stop" signals, or termination codons, marking the end of translation.

17 Fig 17-4. The dictionary of the genetic code.

17 The three bases of an mRNA codon are designated here as the first, second, and third bases, reading in the 5' 3' direction along the mRNA.

17 Practice using this dictionary by finding the codons in FIGURE 17.3

17 The codon AUG not only stands for the amino acid methionine (Met) but also functions as a "start" signal for ribosomes to begin translating the mRNA at that point.

17 Three of the 64 codons function as "stop" signals.

17 Any one of these termination codons marks the end of a genetic message.

17 Notice in FIGURE 17.4 that there is redundancy in the genetic code, but no ambiguity.

17 For example, although codons GAA and GAG both specify glutamic acid (redundancy), neither of them ever specifies any other amino acid (no ambiguity).

17 The redundancy in the code is not altogether random.

17 In many cases, codons that are synonyms for a particular amino acid differ only in the third base of the triplet.

17 We will consider a possible benefit for this redundancy later in the chapter.

17 Our ability to extract the intended message from a written language depends on reading the symbols in the correct groupings--that is, in the correct reading frame.

17 Consider this statement: "The red dog ate the cat."

17 Group the letters incorrectly by starting at the wrong point, and the result will probably be gibberish: for example, "her edd oga tet hec at."

17 The reading frame is also important in the molecular language of cells.

17 The short stretch of polypeptide shown in FIGURE 17.3, for instance, will only be made correctly if the mRNA nucleotides are read from left to right (5' 3') in the groups of three shown in the figure: UGG UUU GGC UCA.

17 Although a genetic message is written with no spaces between the codons, the cell’s protein-synthesizing machinery reads the message as a series of nonoverlapping three-letter words.

17 The message is not read as a series of overlapping words--UGG UUU, and so on--which would convey a very different message.

17 Let’s summarize what we have just covered.

17 Genetic information is encoded as a sequence of nonoverlapping base triplets, or codons, each of which is translated into a specific amino acid during protein synthesis.

17 The genetic code must have evolved very early in the history of life.

17 The genetic code is nearly universal, shared by organisms from the simplest bacteria to the most complex plants and animals.

17 The RNA codon CCG, for instance, is translated as the amino acid proline in all organisms whose genetic code has been examined.

17 In laboratory experiments, genes can be transcribed and translated after they are transplanted from one species to another (FIGURE 17.5).

17 One important application is that bacteria can be programmed by the insertion of human genes to synthesize certain human proteins that have important medical uses.

17 Such applications have produced many exciting developments in biotechnology, which you will learn about in Chapter 20.

17 Fig 17-5. A tobacco plant expressing a firefly gene.

17 Because diverse forms of life share a common genetic code, it is possible to program one species to produce proteins characteristic of another species by transplanting DNA.

17 In this experiment, researchers were able to incorporate a gene from a firefly into the DNA of a tobacco plant.

17 The gene codes for the firefly enzyme that catalyzes the chemical reaction that releases energy in the form of light.

17 Exceptions to the universality of the genetic code are translation systems where a few codons differ from the standard ones.

17 The main examples are found in certain single-celled eukaryotes, such as Paramecium, an organism you may know from the lab.

17 Other examples are found in certain mitochondria and chloroplasts, which transcribe and translate the genes carried by their small amount of DNA.

17 However, the evolutionary significance of the code’s near universality is clear.

17 A language shared by all living things must have been operating very early in the history of life--early enough to be present in the common ancestors of all modern organisms.

17 A shared genetic vocabulary is a reminder of the kinship that bonds all life on Earth.

17 Now that we have considered the linguistic logic and evolutionary significance of the genetic code, we are ready to reexamine transcription, translation, and related topics in more detail.

17 Transcription is the DNA-directed synthesis of RNA: a closer look

17 Messenger RNA, the carrier of information from DNA to the cell’s protein-synthesizing machinery, is transcribed from the template strand of a gene.

17 An enzyme called an RNA polymerase pries the two strands of DNA apart and hooks together the RNA nucleotides as they base-pair along the DNA template (FIGURE 17.6).

17 Like the DNA polymerases that function in DNA replication, RNA polymerases can add nucleotides only to the 3' end of the growing polymer.

17 RNA polymerase moves along a gene from the promoter (green) to just beyond the terminator (red), assembling an RNA molecule (transcript) complementary to the gene’s template strand.

17 In a prokaryote, the RNA transcript of a protein-coding gene is immediately usable as mRNA; in a eukaryote, it must first undergo processing, as described on pp. 311-312.

17 Specific sequences of nucleotides along the DNA mark where transcription of a gene begins and ends.

17 The DNA sequence where RNA polymerase attaches and initiates transcription is known as the promoter; the sequence that signals the end of transcription is called the terminator.

17 Molecular biologists refer to the direction of transcription as "downstream" and the other direction as "upstream."

17 These terms are also used to describe the positions of nucleotide sequences within the DNA or RNA.

17 Thus, the promoter sequence in DNA is said to be upstream from the terminator.

17 The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit.

17 Bacteria have a single type of RNA polymerase that synthesizes not only mRNA but also other types of RNA that function in protein synthesis.

17 In contrast, eukaryotes have three types of RNA polymerase in their nuclei, numbered I, II, and III.

17 The one used for mRNA synthesis is RNA polymerase II.

17 In the discussion of transcription that follows, we start with the features of mRNA synthesis common to both prokaryotes and eukaryotes and then describe some key differences.

17 The three stages of transcription, as shown in FIGURE 17.6 and described next, are initiation, elongation, and termination of the RNA chain.

17 Study FIGURE 17.6 to familiarize yourself with the stages of transcription and the terms used to describe them.

17 RNA Polymerase Binding and Initiation of Transcription

17 The promoter of a gene includes within it the transcription start point (the nucleotide where RNA synthesis actually begins) and typically extends several dozen nucleotide pairs "upstream" from the start point.

17 In addition to serving as a binding site for RNA polymerase and determining where transcription starts, the promoter determines which of the two strands of the DNA helix is used as the template.

17 Certain sections of a promoter are especially important for binding RNA polymerase.

17 In prokaryotes, the RNA polymerase itself specifically recognizes and binds to the promoter.

17 In eukaryotes, a collection of proteins called transcription factors mediate the binding of RNA polymerase and the initiation of transcription.

17 Only after certain transcription factors are attached to the promoter does the RNA polymerase bind to it.

17 The completed assembly of transcription factors and RNA polymerase bound to the promoter is called a transcription initiation complex.

17 FIGURE 17.7 shows the role of transcription factors and a crucial promoter DNA sequence called a TATA box in forming the initiation complex.

17 Fig 17-7. The initiation of transcription at a eukaryotic promoter.

17 In eukaryotic cells, proteins called transcription factors mediate the initiation of transcription by RNA polymerase.

17 The enzyme that transcribes protein-coding genes in eukaryotic cells is called RNA polymerase II.

17 The interaction between eukaryotic RNA polymerase and transcription factors is an example of the special importance of protein-protein interactions in controlling eukaryotic transcription (as we will discuss further in Chapter 19).

17 Once the polymerase is firmly attached to the promoter DNA, the two DNA strands unwind there, and the enzyme starts transcribing the template strand.

17 Elongation of the RNA Strand

17 As RNA polymerase moves along the DNA, it continues to untwist the double helix, exposing about 10 to 20 DNA bases at a time for pairing with RNA nucleotides (see FIGURE 17.6).

17 The enzyme adds nucleotides to the 3' end of the growing RNA molecule as it continues along the double helix.

17 In the wake of this advancing wave of RNA synthesis, the DNA double helix re-forms and the new RNA molecule peels away from its DNA template.

17 Transcription progresses at a rate of about 60 nucleotides per second in eukaryotes.

17 A single gene can be transcribed simultaneously by several molecules of RNA polymerase following each other like trucks in a convoy.

17 A growing strand of RNA trails off from each polymerase, with the length of each new strand reflecting how far along the template the enzyme has traveled from the start point (see FIGURE 17.22).

17 The congregation of many polymerase molecules simultaneously transcribing a single gene increases the amount of mRNA transcribed from it, which helps the cell make the encoded protein in large amounts.

17 Termination of Transcription

17 Transcription proceeds until after the RNA polymerase transcribes a terminator sequence in the DNA.

17 There are several different mechanisms of transcription termination, the details of which are still somewhat murky.

17 In the prokaryotic cell, transcription usually stops right at the end of the termination signal; when the polymerase reaches that point, it releases both the RNA and the DNA.

17 By contrast, in the eukaryotic cell, the polymerase continues for hundreds of nucleotides past the termination signal, which is an AAUAAA sequence in the pre-mRNA (see FIGURE 17.8).

17 But then, at a point about 10 to 35 nucleotides past the AAUAAA, the pre-mRNA is cut free from the enzyme.

17 The cleavage site on the RNA is also the site for the addition of a poly(A) tail--one step of RNA processing, our next topic.

17 Fig 17-8. RNA processing: addition of the 5' cap and poly(A) tail.

17 Enzymes modify the two ends of a eukaryotic pre-mRNA molecule.

17 The modified ends help protect the RNA from degradation, and the poly(A) tail may promote the export of mRNA from the nucleus.

17 When the mRNA reaches the cytoplasm, the modified ends, in conjunction with certain cytoplasmic proteins, facilitate ribosome attachment.

17 The leader and trailer are not translated, nor is the poly(A) tail.

17 Eukaryotic cells modify RNA after transcription

17 Enzymes in the eukaryotic nucleus modify pre-mRNA in various ways before the genetic messages are dispatched to the cytoplasm.

17 During this RNA processing, both ends of the primary transcript are usually altered.

17 In most cases, certain interior sections of the molecule are then cut out and the remaining parts spliced together.

17 Alteration of mRNA Ends

17 Each end of a pre-mRNA molecule is modified in a particular way.

17 The 5' end, the end made first during transcription, is immediately capped off with a modified form of a guanine (G) nucleotide.

17 This 5' cap has at least two important functions.

17 First, it helps protect the mRNA from degradation by hydrolytic enzymes.

17 Second, after the mRNA reaches the cytoplasm, the 5' cap functions as part of an "attach here" sign for ribosomes.

17 The other end of an mRNA molecule, the 3' end, is also modified before the message exits the nucleus.

17 At the 3' end, an enzyme makes a poly(A) tail consisting of some 50 to 250 adenine nucleotides.

17 Like the 5' cap, the poly(A) tail inhibits degradation of the RNA and probably helps ribosomes attach to it.

17 The poly(A) tail also seems to facilitate the export of mRNA from the nucleus.

17 FIGURE 17.8 shows a eukaryotic mRNA molecule with cap and tail; it also shows the nontranslated leader and trailer segments of RNA to which they are attached.

17 Split Genes and RNA Splicing\ \ The most remarkable stage of RNA processing in the eukaryotic nucleus is the removal of a large portion of the RNA molecule that is initially synthesized--a cut-and-paste job called RNA splicing (FIGURE 17.9, p. 312).

17 The average length of a transcription unit along a eukaryotic DNA molecule is about 8,000 nucleotides, so the primary RNA transcript is also that long.

17 But it takes only about 1,200 nucleotides to code for an average-sized protein of 400 amino acids.

17 Remember, each amino acid is encoded by a triplet of nucleotides.

17 This means that most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides, regions that are not translated.

17 Even more surprising is that most of these noncoding sequences are interspersed between coding segments of the gene and thus between coding segments of the pre-mRNA.

17 In other words, the sequence of DNA nucleotides that codes for a eukaryotic polypeptide is not continuous.

17 The noncoding segments of nucleic acid that lie between coding regions are called intervening sequences, or introns for short.

17 The other regions are called exons, because they are eventually expressed, usually by being translated into amino acid sequences.

17 Exceptions include the leader and trailer portions of the exons at the ends of the RNA.

17 Because of these exceptions, you may find it helpful to think of exons as DNA that exits the nucleus.)

17 The terms intron and exon are used for both DNA and RNA.

17 Richard Roberts and Phillip Sharp, who independently found evidence of "split genes" in 1977, shared a Nobel Prize in 1993 for this discovery.

17 Fig 17-9. RNA processing: RNA splicing.

17 The RNA molecule shown here codes for b globin, one of the polypeptides of hemoglobin.

17 The numbers under the RNA refer to codons; b globin is 146 amino acids long.

17 The b globin gene and its pre-mRNA transcript have three regions, called exons, that consist mostly of coding sequences; exons are separated by noncoding regions, called introns.

17 During RNA processing, the introns are excised and the exons are spliced together.

17 In making a primary transcript from a gene, RNA polymerase transcribes both introns and exons from the DNA, but the mRNA molecule that enters the cytoplasm is an abridged version.

17 The introns are cut out from the molecule and the exons joined together to form an mRNA molecule with a continuous coding sequence.

17 This is the process of RNA splicing.

17 How is pre-mRNA splicing carried out?

17 Researchers have learned that the signals for RNA splicing are short nucleotide sequences at the ends of introns.

17 At least some introns contain sequences that control gene activity in some way, and the splicing process itself may help regulate the passage of mRNA from nucleus to cytoplasm.

17 One established benefit of split genes is to enable a single gene to encode more than one kind of polypeptide.

17 A number of genes are known to give rise to two or more different polypeptides, depending on which segments are treated as exons during RNA processing; this is called alternative RNA splicing (see FIGURE 19.11).

17 The fruit fly provides an interesting example: Sex differences in this animal are largely due to differences in how males and females splice the RNA transcribed from certain genes.

17 Early results from the Human Genome Project (discussed in Chapter 20) suggest that alternative RNA splicing may be one reason humans can get along with a relatively small number of genes--only about twice as many as a fruit fly.

17 Split genes may also facilitate the evolution of new and potentially useful proteins.

17 Proteins often have a modular architecture consisting of discrete structural and functional regions called domains.

17 One domain of an enzymatic protein, for instance, might include the active site, while another might attach the protein to a cellular membrane.

17 In many cases, different exons code for the different domains of a protein (FIGURE 17.11).

17 Introns increase the probability of potentially beneficial crossing over between genes.

17 Simply by providing more places where crossing over can occur (and without interfering with coding sequences), introns increase the opportunity for recombination between two alleles of a gene and raise the probability that a crossover will switch one version of an exon for another version found on the homologous chromosome.

17 We can also imagine the occasional mixing and matching of exons between completely different (nonallelic) genes.

17 Exon shuffling of either sort could lead to new proteins with novel combinations of functions.

17 Fig 17-11. Correspondence between exons and protein domains.

17 In a number of genes, different exons encode separate domains of the protein product.

17 Each domain, an independently folding part of the protein, performs a different function.

17 Such correspondence between exons and domains suggests that new proteins can evolve by exon shuffling among genes.

17 THE SYNTHESIS OF PROTEIN\ \ Translation is the RNA-directed synthesis of a polypeptide: a closer look

17 Signal peptides target some eukaryotic polypeptides to specific destinations in the cell

17 RNA plays multiple roles in the cell: a review

17 Comparing protein synthesis in prokaryotes and eukaryotes: a review

17 Point mutations can affect protein structure and function

17 What is a gene? revisiting the question

17 We will now examine more closely how genetic information flows from mRNA to protein--the process of translation.

17 As we did for transcription, we’ll concentrate on the basic steps of translation that occur in both prokaryotes and eukaryotes while pointing out key differences.

17 Translation is the RNA-directed synthesis of a polypeptide: a closer look

17 In the process of translation, a cell interprets a genetic message and builds a protein accordingly.

17 The message is a series of codons along an mRNA molecule, and the interpreter is called transfer RNA (tRNA).

17 The function of tRNA is to transfer amino acids from the cytoplasm’s amino acid pool to a ribosome.

17 A cell keeps its cytoplasm stocked with all 20 amino acids, either by synthesizing them from other compounds or by taking them up from the surrounding solution.

17 The ribosome adds each amino acid brought to it by tRNA to the growing end of a polypeptide chain (FIGURE 17.12, p. 314).

17 Fig 17-12. Translation: the basic concept.

17 As a molecule of mRNA is moved through a ribosome, codons are translated into amino acids, one by one.

17 The interpreters are tRNA molecules, each type with a specific anticodon at one end and a certain amino acid at the other end.

17 A tRNA adds its amino acid cargo to a growing polypeptide chain when the anticodon bonds to a complementary codon on the mRNA.

17 The figures that follow show some of the details of translation in the prokaryotic cell.

17 Molecules of tRNA are not all identical.

17 The key to translating a genetic message into a specific amino acid sequence is that each type of tRNA molecule links a particular mRNA codon with a particular amino acid.

17 As a tRNA molecule arrives at a ribosome, it bears a specific amino acid at one end.

17 At the other end is a nucleotide triplet called an anticodon, which base-pairs with a complementary codon on mRNA.

17 For example, consider the mRNA codon UUU, which is translated as the amino acid phenylalanine (see FIGURE 17.4).

17 The tRNA that plugs into this codon by hydrogen bonding has AAA as its anticodon and carries phenylalanine at its other end.

17 As an mRNA molecule is moved through a ribosome, phenylalanine will be added to the polypeptide chain whenever the codon UUU is presented for translation.

17 Codon by codon, the genetic message is translated as tRNAs deposit amino acids in the order prescribed, and the ribosome joins the amino acids into a chain.

17 The tRNA molecule is like a flash card with a nucleic acid word (anticodon) on one side and a protein word (amino acid) on the other.

17 Translation is simple in principle but complex in its biochemistry and mechanics, especially in the eukaryotic cell.

17 In dissecting translation, we’ll concentrate on the slightly less complicated version of the process that occurs in prokaryotes.

17 Let’s first look at some of the major players in this cellular drama, then see how they act together to make a polypeptide.

17 The Structure and Function of Transfer RNA

17 Like mRNA and other types of cellular RNA, transfer RNA molecules are transcribed from DNA templates.

17 In a eukaryotic cell, tRNA, like mRNA, is made in the nucleus and must travel from the nucleus to the cytoplasm, where translation occurs.

17 In both prokaryotic and eukaryotic cells, each tRNA molecule is used repeatedly, picking up its designated amino acid in the cytosol, depositing this cargo at the ribosome, and then leaving the ribosome to pick up another load.

17 As illustrated in FIGURE 17.13, a tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long (compared to hundreds of nucleotides for most mRNA molecules).

17 This RNA strand folds back upon itself to form a molecule with a three-dimensional structure reinforced by interactions between different parts of the nucleotide chain.

17 Nucleotide bases in certain regions of the tRNA strand form hydrogen bonds with complementary bases of other regions.

17 Flattened into one plane to reveal this base pairing, a tRNA molecule looks like a cloverleaf.

17 The tRNA actually twists and folds into a compact three-dimensional structure that is roughly L-shaped.

17 The loop protruding from one end of the L includes the anticodon, the specialized base triplet that binds to a specific mRNA codon.

17 From the other end of the L-shaped tRNA molecule protrudes its 3' end, which is the attachment site for an amino acid.

17 A ribosome, which is large enough to be seen with an electron microscope, is made up of two subunits, called the large and small subunits (FIGURE 17.15).

17 The ribosomal subunits are constructed of proteins and RNA molecules named ribosomal RNA (rRNA).

17 In eukaryotes, the subunits are made in the nucleolus.

17 Ribosomal RNA genes on the chromosomal DNA are transcribed, and the RNA is processed and assembled with proteins imported from the cytoplasm.

17 The resulting ribosomal subunits are then exported via nuclear pores to the cytoplasm.

17 In both prokaryotes and eukaryotes, large and small subunits join to form a functional ribosome only when they attach to an mRNA molecule.

17 About two-thirds of the mass of a ribosome is rRNA.

17 Because most cells contain thousands of ribosomes, rRNA is the most abundant type of RNA.

17 Fig 17-15. The anatomy of a functioning ribosome.

17 Although the ribosomes of prokaryotes and eukaryotes are very similar in structure and function, those of eukaryotes are slightly larger and differ somewhat from prokaryotic ribosomes in their molecular composition.

17 The differences are medically significant.

17 Certain antibiotic drugs can paralyze prokaryotic ribosomes without inhibiting the ability of eukaryotic ribosomes to make proteins.

17 These drugs, including tetracycline and streptomycin, are used to combat bacterial infections.

17 The structure of a ribosome reflects its function of bringing mRNA together with amino acid-bearing tRNAs.

17 In addition to a binding site for mRNA, each ribosome has three binding sites for tRNA (see FIGURE 17.15).

17 The P site (peptidyl-tRNA site) holds the tRNA carrying the growing polypeptide chain, while the A site (aminoacyl-tRNA site) holds the tRNA carrying the next amino acid to be added to the chain.

17 Discharged tRNAs leave the ribosome from the E site (exit site).

17 The ribosome holds the tRNA and mRNA close together and positions the new amino acid for addition to the carboxyl end of the growing polypeptide.

17 It then catalyzes the formation of the peptide bond.

17 Four decades of genetic and biochemical research on ribosome structure recently culminated in the detailed structure of the bacterial ribosome.

17 One view of the large subunit is shown in FIGURE 17.16, and a ribbon model of the entire ribosome appears at the beginning of this chapter.

17 Ribosome structure strongly supports the hypothesis that rRNA, not protein, carries out the ribosome’s functions.

17 RNA is the main constituent of the interface between the two subunits and of the A and P sites, and it is the catalyst of peptide bond formation.

17 Thus, a ribosome can be regarded as one colossal ribozyme!

17 The ribosome’s proteins are largely on the exterior, apparently playing a mainly structural role.

17 Fig 17-16. Structure of the large ribosomal subunit at the atomic level.

17 In 2000, researchers succeeded in determining the atomic structures of both subunits of the bacterial ribosome using X-ray crystallography (see FIGURE 5.27).

17 This computer model shows the large subunit from a point of view different from that in FIGURE 17.15.

17 Here we are looking at the "bottom" of the large subunit, as it would look from an attached small subunit.

17 The rRNA molecules are colored orange or maroon (sugar-phosphate backbones) and gray (bases) and the proteins are purple.

17 Notice that the proteins are mostly on the outside of the subunit, whereas the rRNA, responsible for the ribosome’s functions, is mostly in the interior.

17 The tRNA molecules are included for orientation.

17 Building a Polypeptide

17 We can divide translation, the synthesis of a polypeptide chain, into three stages (analogous to those of transcription): initiation, elongation, and termination.

17 All three stages require protein "factors" that aid mRNA, tRNA, and ribosomes in the translation process.

17 For chain initiation and elongation, energy is also required.

17 It is provided by the hydrolysis of GTP (guanosine triphosphate), a molecule closely related to ATP.

17 Initiation. \ \ The initiation stage of translation brings together mRNA, a tRNA bearing the first amino acid of the polypeptide, and the two subunits of a ribosome (FIGURE 17.17).

17 First, a small ribosomal subunit binds to both mRNA and a special initiator tRNA.

17 The small ribosomal subunit attaches to the leader segment at the 5' (upstream) end of the mRNA.

17 In bacteria, rRNA of the small subunit base-pairs with a specific sequence of nucleotides within the mRNA leader; in eukaryotes, the 5' cap first tells the small subunit to attach to the 5' end of the mRNA.

17 Downstream on the mRNA is the initiation codon, AUG, which signals the start of translation.

17 The initiator tRNA, which carries the amino acid methionine, attaches to the initiation codon.

17 Fig 17-17. The initiation of translation.

17 The union of mRNA, initiator tRNA, and a small ribosomal subunit is followed by the attachment of a large ribosomal subunit, completing a translation initiation complex.

17 Proteins called initiation factors are required to bring all these components together.

17 The cell also spends energy in the form of a GTP molecule to form the initiation complex.

17 At the completion of the initiation process, the initiator tRNA sits in the P site of the ribosome, and the vacant A site is ready for the next aminoacyl tRNA.

17 The synthesis of a polypeptide is initiated at its amino end (see FIGURE 5.16b).

17 Elongation. \ \ In the elongation stage of translation, amino acids are added one by one to the preceding amino acid.

17 Each addition involves the participation of several proteins called elongation factors and occurs in a three-step cycle (FIGURE 17.18):

17 Fig 17-18. The elongation cycle of translation.

17 Not shown in this diagram are the proteins called elongation factors.

17 The hydrolysis of GTP drives the elongation process.

17 1. Codon recognition.\ \ The mRNA codon in the A site of the ribosome forms hydrogen bonds with the anticodon of an incoming molecule of tRNA carrying its appropriate amino acid.

17 An elongation factor ushers the tRNA into the A site.

17 This step requires the hydrolysis of two molecules of GTP.

17 2. Peptide bond formation.\ \ An rRNA molecule of the large ribosomal subunit, functioning as a ribozyme, catalyzes the formation of a peptide bond that joins the polypeptide extending from the P site to the newly arrived amino acid in the A site.

17 In this step, the polypeptide separates from the tRNA to which it was attached, and the amino acid at its carboxyl end bonds to the amino acid carried by the tRNA in the A site.

17 3. Translocation.\ \ The ribosome now translocates (moves) the tRNA in the A site, with its attached polypeptide, to the P site.

17 As the tRNA moves, its anticodon remains hydrogen-bonded to the mRNA codon; the mRNA moves along with it and brings the next codon to be translated into the A site.

17 Meanwhile, the tRNA that was in the P site is moved to the E (exit) site and from there leaves the ribosome.

17 The translocation step requires energy, which is provided by hydrolysis of a GTP molecule.

17 The mRNA is moved through the ribosome in one direction only, 5' end first; this is equivalent to the ribosome moving 5' 3'on the mRNA.

17 The important point is that the ribosome and the mRNA move relative to each other, unidirectionally, codon by codon.

17 The elongation cycle takes less than a tenth of a second and is repeated as each amino acid is added to the chain until the polypeptide is completed.

17 Termination. \ \ The final stage of translation is termination (FIGURE 17.19).

17 Elongation continues until a stop codon in the mRNA reaches the A site of the ribosome.

17 The special base triplets UAA, UAG, and UGA do not code for amino acids but instead act as signals to stop translation.

17 A protein called a release factor binds directly to the stop codon in the A site.

17 The release factor causes the addition of a water molecule instead of an amino acid to the polypeptide chain.

17 This reaction hydrolyzes the completed polypeptide from the tRNA that is in the P site, freeing the polypeptide from the ribosome.

17 The remainder of the translation assembly then comes apart.

17 Fig 17-19. The termination of translation.

17 Polyribosomes\ \ A single ribosome can make an average-sized polypeptide in less than a minute.

17 Typically, however, a single mRNA is used to make many copies of a polypeptide simultaneously, because a number of ribosomes work on translating the message at the same time.

17 Once a ribosome moves past the initiation codon, a second ribosome can attach to the mRNA, and thus, multiple ribosomes may trail along the same mRNA.

17 Such strings of ribosomes, called polyribosomes, can be seen with the electron microscope (FIGURE 17.20).

17 Polyribosomes are found in both prokaryotic and eukaryotic cells.

17 They help a cell to make many copies of a polypeptide very quickly.

17 Fig 17-20. Polyribosomes.

17 From Polypeptide to Functional Protein

17 During and after its synthesis, a polypeptide chain begins to coil and fold spontaneously, forming a functional protein of specific conformation: a three-dimensional molecule with secondary and tertiary structure (see FIGURE 5.24).

17 Additional steps--posttranslational modifications--may be required before the protein can begin doing its particular job in the cell.

17 Certain amino acids may be chemically modified by the attachment of sugars, lipids, phosphate groups, or other additions.

17 Enzymes may remove one or more amino acids from the leading (amino) end of the polypeptide chain.

17 In some cases, a single polypeptide chain may be enzymatically cleaved into two or more pieces.

17 For example, the protein insulin is first synthesized as a single polypeptide chain but becomes active only after an enzyme cuts out a central part of the chain, leaving a protein made up of two polypeptide chains connected by disulfide bridges (see FIGURE 5.22).

17 In other cases, two or more polypeptides that are synthesized separately may join to become the subunits of a protein that has quaternary structure.